VAMP-associated protein A-interacting proteins and use thereof

ABSTRACT

Protein complexes are provided comprising VAP-A and one or more VAP-A-interacting proteins. The protein complexes are useful in screening assays for identifying compounds effective in modulating the protein complexes and in treating and/or preventing diseases and disorders associated with VAP-A and its interacting partners. In addition, methods of detecting the protein complexes and modulating the functions and activities of the protein complexes or interacting members thereof are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claim priority under 35 U.S.C. §119(e) to provisionalapplication Ser. No. 60/291,730, filed May 17, 2001, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to protein-protein interactions,particularly to protein complexes formed by protein-protein interactionsand methods of use thereof.

BACKGROUND OF THE INVENTION

The prolific output from numerous genomic sequencing efforts, includingthe Human Genome Project, is creating an ever-expanding foundation forlarge-scale study of protein function. Indeed, this emerging field ofproteomics can appropriately be viewed as a bridge that connects DNAsequence information to the physiology and pathology of intactorganisms. As such, proteomics—the large-scale study of proteinfunction—will likely be starting point for the development of manyfuture pharmaceuticals. The efficiency of drug development willtherefore depend on the diversity and robustness of the methods used toelucidate protein function, i.e., the proteomic tools that areavailable.

Several approaches are generally known in the art for studying proteinfunction. One method is to analyze the DNA sequence of a particular geneand the amino acid sequence coded by the gene in the context ofsequences of genes with known functions. Generally, similar functionscan be predicted based on sequence homologies. This “homology method”has been widely used, and powerful computer programs have been designedto facilitate homology analysis. See, e.g., Altschul et al., NucleicAcids Res., 25:3389-3402 (1997). However, this method is useful onlywhen the function of a homologous protein is known.

Another useful approach is to interfere with the expression of aparticular gene in a cell or organism and examine the consequentphenotypic effects. For example, Fire et al., Nature, 391:806-811 (1998)disclose an “RNA interference” assay in which double-stranded RNAtranscripts corresponding to a particular target gene are injected intocells or organisms to determine the phenotype associated with thedisrupted expression of that gene. Alternatively, transgenictechnologies can be utilized to delete or “knock out” a particular genein an organism and the effect of the gene knockout is determined. Seee.g., Winzeler et al., Science, 285:901-906 (1999); Zambrowicz et al.,Nature, 392:608-611 (1998). The phenotypic effects resulting from thedisruption of expression of a particular gene can shed some light on thefunctions of the gene. However, the techniques involved are complex andthe time required for a phenotype to appear can be long, especially inmammals. In addition, in many cases disruption of a particular gene maynot cause any detectable phenotypic effect.

Gene functions can also be uncovered by genetic linkage analysis. Forexample, genes responsible for certain diseases may be identified bypositional cloning. Alternatively, gene function may be inferred bycomparing genetic variations among individuals in a population andcorrelating particular phenotypes with the genetic variations. Suchlinkage analyses are powerful tools, particularly when geneticvariations exist in a traceable population from which samples arereadily obtainable. However, readily identifiable genetic diseases arerare and samples from a large population with genetic variations are noteasily accessible. In addition, it is also possible that a geneidentified in a linkage analysis does not contribute to the associateddisease or symptom but rather is simply linked to unknown geneticvariations that cause the phenotypic defects.

With the advance of bioinformatics and publication of the full genomesequence of many organisms, computational methods have also beendeveloped to assign protein functions by comparative genome analysis.For example, Pellegrini et al., Proc. Natl. Acad. Sci. USA 96:4285-4288(1999) discloses a method that constructs a “phylogenetic profile” thatsummarizes the presence or absence of a particular protein across anumber of organisms as determined by analyzing the genome sequences ofthe organisms. A protein's function is predicted to be linked to anotherprotein's function if the two proteins share the same phylogeneticprofile. Another method, the Rosetta Stone method, is based on thetheory that separate proteins in one organism are often expressed asseparate domains of a fusion protein in another organism. Because theseparate domains in the fusion protein are predictably associated withthe same function, it can be reasonably predicted that the separateproteins are associated with same functions. Therefore, by discoveringseparate proteins corresponding to a fusion protein, i.e., the “RosettaStone sequence,” functional linkage between proteins can be established.See Marcotte et al., Science, 285:751-753 (1999); Enright et al.,Nature, 402:86-90 (1999). Another computational method is the “geneneighbor method.” See Dandekar et al., Trends Biochem. Sci., 23:324-328(1998); Overbeek et al., Proc. Natl. Acad. Sci. USA 96:2896-2901 (1999).This method is based on the likelihood that if two genes are found to beneighbors in several different genomes, the proteins encoded by thegenes share a common function.

While the methods described above are useful in analyzing proteinfunctions, they are constrained by various practical limitations such asunavailability of suitable samples, inefficient assay procedures, andlimited reliability. The computational methods are useful in linkingproteins by function. However, they are only applicable to certainproteins, and the linkage maps established therewith are sketchy. Thatis, the maps lack specific information that describes how proteinsfunction in relation to each other within the functional network.Indeed, none of the methods places the identified protein functions inthe context of protein-protein interactions.

In contrast with the traditional view of protein function, which focuseson the action of a single protein molecule, a modem expanded view ofprotein function defines a protein as an element in an interactionnetwork. See Eisenberg et al., Nature, 405:823-826 (2000). That is, afull understanding of the functions of a protein will require knowledgeof not only the characteristics of the protein itself, but also itsinteractions or connections with other proteins in the same interactingnetwork. In essence, protein-protein interactions form the basis ofalmost all biological processes, and each biological process is composedof a network of interacting proteins. For example, cellular structuressuch as cytoskeletons, nuclear pores, centrosomes, and kinetochores areformed by complex interactions among a multitude of proteins. Manyenzymatic reactions are associated with large protein complexes formedby interactions among enzymes, protein substrates, and proteinmodulators. In addition, protein-protein interactions are also part ofthe mechanisms for signal transduction and other basic cellularfunctions such as DNA replication, transcription, and translation. Forexample, the complex transcription initiation process generally requiresprotein-protein interactions among numerous transcription factors, RNApolymerase, and other proteins. See e.g., Tjian and Maniatis, Cell,77:5-8 (1994).

Because most proteins function through their interactions with otherproteins, if a test protein interacts with a known protein, one canreasonably predict that the test protein is associated with thefunctions of the known protein, e.g., in the same cellular structure orsame cellular process as the known protein. Thus, interaction partnerscan provide an immediate and reliable understanding towards thefunctions of the interacting proteins. By identifying interactingproteins, a better understanding of disease pathways and the cellularprocesses that result in diseases may be achieved, and importantregulators and potential drug targets in disease pathways can beidentified.

There has been much interest in protein-protein interactions in thefield of proteomics. A number of biochemical approaches have been usedto identify interacting proteins. These approaches generally employ theaffinities between interacting proteins to isolate proteins in a boundstate. Examples of such methods include coimmunoprecipitation andcopurification, optionally combined with cross-linking to stabilize thebinding. Identities of the isolated protein interacting partners can becharacterized by, e.g., mass spectrometry. See e.g., Rout et al., J.Cell. Biol., 148:635-651 (2000); Houry et al., Nature, 402:147-154(1999); Winter et al., Curr. Biol., 7:517-529 (1997). A popular approachuseful in large-scale screening is the phage display method, in whichfilamentous bacteriophage particles are made by recombinant DNAtechnologies to express a peptide or protein of interest fused to acapsid or coat protein of the bacteriophage. A whole library of peptidesor proteins of interest can be expressed and a bait protein can be usedto screening the library to identify peptides or proteins capable ofbinding to the bait protein. See e.g., U.S. Pat. Nos. 5,223,409;5,403,484; 5,571,698; and 5,837,500. Notably, the phage display methodonly identifies those proteins capable of interacting in an in vitroenvironment, while the coimmunoprecipitation and copurification methodsare not amenable to high throughput screening.

The yeast two-hybrid system is a genetic method that overcomes certainshortcomings of the above approaches. The yeast two-hybrid system hasproven to be a powerful method for the discovery of specific proteininteractions in vivo. See generally, Bartel and Fields, eds., The YeastTwo-Hybrid System, Oxford University Press, New York, N.Y., 1997. Theyeast two-hybrid technique is based on the fact that the DNA-bindingdomain and the transcriptional activation domain of a transcriptionalactivator contained in different fusion proteins can still activate genetranscription when they are brought into proximity to each other. In ayeast two-hybrid system, two fusion proteins are expressed in yeastcells. One has a DNA-binding domain of a transcriptional activator fusedto a test protein. The other, on the other hand, includes atranscriptional activating domain of the transcriptional activator fusedto another test protein. If the two test proteins interact with eachother in vivo, the two domains of the transcriptional activator arebrought together reconstituting the transcriptional activator andactivating a reporter gene controlled by the transcriptional activator.See, e.g., U.S. Pat. No. 5,283,173.

Because of its simplicity, efficiency and reliability, the yeasttwo-hybrid system has gained tremendous popularity in many areas ofresearch. In addition, yeast cells are eukaryotic cells. Theinteractions between mammalian proteins detected in the yeast two-hybridsystem typically are bona fide interactions that occur in mammaliancells under physiological conditions. As a matter of fact, numerousmammalian protein-protein interactions have been identified using theyeast two-hybrid system. The identified proteins have contributedsignificantly to the understanding of many signal transduction pathwaysand other biological processes. For example, the yeast two-hybrid systemhas been successfully employed in identifying a large number of novelmammalian cell cycle regulators that are important in complex cell cycleregulations. Using known proteins that are important in cell cycleregulation as baits, other proteins involved in cell cycle control wereidentified by virtue of their ability to interact with the baits. Seegenerally, Hannon et al., in The Yeast Two-Hybrid System, Bartel andFields, eds., pages 183-196, Oxford University Press, New York, N.Y.,1997. Examples of mammalian cell cycle regulators identified by theyeast two-hybrid system include CDK4/CDK6 inhibitors (e.g., p16, p15,p18 and p19), Rb family members (e.g., p130), Rb phosphatase (e.g.,PP1-α2), Rb-binding transcription factors (e.g., E2F-4 and E2F-5),General CDK inhibitors (e.g., p21 and p27), CAK cyclin (e.g., cyclin H),and CDK Thr161 phosphatase (e.g., KAP and CDI1). See id at page 192.“[T]he two-hybrid approach promises to be a useful tool in our ongoingquest for new pieces of the cell cycle puzzle.” See id at page 193.

The yeast two-hybrid system can be employed to identify proteins thatinteract with a specific known protein involved in a disease pathway,and thus provide valuable understandings of the disease mechanism. Theidentified proteins and the protein-protein interactions in which theyparticipate are potential targets for use in identifying new drugs fortreating the disease.

SUMMARY OF THE INVENTION

It has been discovered that VAMP-associated protein A (VAP-A)specifically interacts with protein phosphatase, type 1,glycogen-binding, regulatory subunit 3 (PPP1R3) and glucosetransporter-like protein III (GTR3). The specific interactions betweenthese proteins and VAP-A suggest that VAP-A and the VAP-A-interactingproteins are involved in common biological processes. In addition, theinteractions between such VAP-A-interacting proteins and VAP-A lead tothe formation of protein complexes both in vitro and in vivo thatcontain VAP-A and one or more of the VAP-A-interacting proteins. Theprotein complexes formed under physiological conditions can mediate thefunctions and biological activities of VAP-A and the VAP-A-interactingproteins. For example, they may be involved in docking and fusion ofmembrane vesicles to target organelle membranes and cellular uptake ofglucose. Thus, the VAP-A-interacting proteins and the protein complexesare potential drug targets for the development of drugs useful intreating or preventing diseases and disorders associated with theVAP-A-containing protein complexes or a protein member thereof.

In accordance with a first aspect of the present invention, isolatedprotein complexes are provided comprising VAP-A and one or moreVAP-A-interacting proteins selected from the group consisting of PPP1R3and GTR3. In addition, homologues, derivatives, and fragments of VAP-Aand of the VAP-A-interacting proteins may also be used in formingprotein complexes. In a specific embodiment, fragments of VAP-A and theVAP-A-interacting proteins containing the protein domains responsiblefor the interaction between VAP-A and the VAP-A-interacting proteins areused in forming a protein complex of the present invention. In anotherembodiment, an interacting protein member in the protein complexes ofthe present invention is a fusion protein containing VAP-A or ahomologue, derivative, or fragment thereof. A fusion protein containinga VAP-A-interacting protein or a homologue, derivative, or fragmentthereof may also be used in the protein complexes. In yet anotherembodiment, a protein complex is provided from a hybrid protein, whichcomprises VAP-A or a homologue, derivative, or fragment thereofcovalently linked, directly or through a linker, to one of theVAP-A-interacting proteins or a homologue, derivative, or fragmentthereof. In addition, nucleic acids encoding the hybrid protein are alsoprovided.

In yet another aspect, the present invention also provides a method formaking the protein complexes. The method includes the steps of providingthe first protein and the second protein in the protein complexes of thepresent invention and contacting said first protein with said secondprotein. In addition, the protein complexes can be prepared by isolationor purification from tissues and cells or produced by recombinantexpression of their protein members. The protein complexes can beincorporated into a protein microchip or microarray, which are useful inlarge-scale high throughput screening assays involving the proteincomplexes.

In accordance with a second aspect of the invention, antibodies areprovided that are immunoreactive with a protein complex of the presentinvention. In one embodiment, an antibody is selectively immunoreactivewith a protein complex of the present invention. In another embodiment,a bifunctional antibody is provided that has two different antigenbinding sites, each being specific to a different interacting proteinmember in a protein complex of the present invention. The antibodies ofthe present invention can take various forms including polyclonalantibodies, monoclonal antibodies, chimeric antibodies, antibodyfragments such as Fv fragments, single-chain Fv fragments (scFv), Fab′fragments, and F(ab′)₂ fragments. Preferably, the antibodies arepartially or fully humanized antibodies. The antibodies of the presentinvention can be readily prepared using procedures generally known inthe art. For example, recombinant libraries such as phage displaylibraries and ribosome display libraries may be used to screen forantibodies with desirable specificities. In addition, variousmutagenesis techniques such as site-directed mutagenesis and PCRdiversification may be used in combination with the screening assays.

The present invention also provides detection methods for determiningwhether there is any aberration in a patient with respect to a proteincomplex having VAP-A and one or more of the VAP-A-interacting proteins.In one embodiment, the method comprises detecting an aberrantconcentration of the protein complexes of the present invention.Alternatively, the concentrations of one or more interacting proteinmembers (at the protein or CDNA or mRNA level) of a protein complex ofthe present invention are measured. In addition, the cellularlocalization, or tissue or organ distribution of a protein complex ofthe present invention is determined to detect any aberrant localizationor distribution of the protein complex. In another embodiment, mutationsin one or more interacting protein members of a protein complex of thepresent invention can be detected. In particular, it is desirable todetermine whether the interacting protein members have any mutationsthat will lead to, or are associated with, changes in the functionalactivity of the proteins or changes in their binding affinity to otherinteracting protein members in forming a protein complex of the presentinvention. In yet another embodiment, the binding constant of theinteracting protein members of one or more protein complexes isdetermined. A kit may be used for conducting the detection methods ofthe present invention. Typically, the kit contains reagents useful inany of the above-described embodiments of the detection methods,including, e.g., antibodies specific to a protein complex of the presentinvention or interacting members thereof, and oligonucleotidesselectively hybridizable to the cDNAs or mRNAs encoding one or moreinteracting protein members of a protein complex. The detection methodsmay be useful in diagnosing a disease or disorder such as diabetes,obesity, ischemia, and insulin resistance, staging the disease ordisorder, or identifying a predisposition to the disease or disorder.

The present invention also provides screening methods for selectingmodulators of a protein complex formed between VAP-A or a homologue,derivative or fragment thereof and one of the VAP-A-interacting proteinsor a homologue, derivative, or fragment thereof. Screening methods arealso provided for selecting modulators of VAP-A or a VAP-A-interactingprotein. The compounds identified in the screening methods of thepresent invention can be used in modulating the functions or activitiesof VAP-A, the VAP-A-interacting proteins, or the protein complexes ofthe present invention. They may also be effective in modulating thecellular functions involving VAP-A, VAP-A-interacting proteins orVAP-A-containing protein complexes, and in preventing or amelioratingdiseases or disorders such as diabetes, obesity, ischemia, and insulinresistance.

Thus, test compounds may be screened in in vitro binding assays toidentify compounds capable of binding a protein complex of the presentinvention, or VAP-A or a VAP-A-interacting protein identified inaccordance with the present invention or homologues, derivatives orfragments thereof. The assays may include the steps of contacting theprotein complex with a test compound and detecting the interactionbetween the interacting partners. In addition, in vitro dissociationassays may also be employed to select compounds capable of dissociatingor destabilizing the protein complexes identified in accordance with thepresent invention. For example, the assays may entail (1) contacting theinteracting members of the protein complex with each other in thepresence of a test compound; and (2) detecting the interaction betweenthe interacting members. An in vitro screening assay may also be used toidentify compounds that trigger or initiate the formation of, orstabilize, a protein complex of the present invention.

In preferred embodiments, in vivo assays such as yeast two-hybrid assaysand various derivatives thereof, preferably reverse two-hybrid assays,are utilized in identifying compounds that interfere with or disruptprotein-protein interactions between VAP-A or a homologue, derivative orfragment thereof and a VAP-A-interacting protein or a homologue,derivative or fragment thereof. In addition, systems such as yeasttwo-hybrid assays are also useful in selecting compounds capable oftriggering or initiating, enhancing or stabilizing protein-proteininteractions between VAP-A or a homologue, derivative or fragmentthereof and a VAP-A-interacting protein of the present invention or ahomologue, derivative or fragment thereof.

In a specific embodiment, the screening method includes: (a) providingin a host cell a first fusion protein having a first protein which isVAP-A or a homologue or derivative or fragment thereof, and a secondfusion protein having a second protein which is VAP-A-interactingprotein as provided in the present invention, or a homologue orderivative or fragment thereof, wherein a DNA binding domain is fused toone of the first and second proteins while a transcription-activatingdomain is fused to the other of said first and second proteins; (b)providing in the host cell a reporter gene, wherein the transcription ofthe reporter gene is determined by the interaction between the firstprotein and the second protein; (c) allowing the first and second fusionproteins to interact with each other within the host cell in thepresence of a test compound; and (d) determining the presence or absenceof expression of the reporter gene.

In addition, the present invention also provides a method for selectinga compound capable of modulating a protein-protein interaction betweenVAP-A and a VAP-A-interacting protein in a protein complex, whichcomprises the steps of (1) contacting a test compound with aVAP-A-interacting protein or a homologue or derivative or fragmentthereof, and (2) determining whether said test compound is capable ofbinding said protein. In a preferred embodiment, the method furtherincludes testing a selected test compound capable of binding saidprotein for its ability to interfere with a protein-protein interactionbetween VAP-A and the VAP-A-interacting protein, and optionally furthertesting the selected test compound capable of binding said protein forits ability to modulate cellular activities associated with VAP-A and/orthe VAP-A-interacting protein.

The present invention also relates to a virtual screen method forproviding a compound capable of modulating an interaction between theinteracting members in the protein complexes of the present invention.In one embodiment, the method comprises the steps of providing atomiccoordinates defining a three-dimensional structure of a protein complexof the present invention, and designing or selecting compounds capableof interfering with the interaction between said first protein and saidsecond protein based on said atomic coordinates. In another embodiment,the method comprises the steps of providing atomic coordinates defininga three-dimensional structure of VAP-A, or a VAP-A-interacting protein,and designing or selecting compounds capable of binding VAP-A or theVAP-A-interacting protein based on said atomic coordinates. In preferredembodiments, the method further includes testing a selected testcompound for its ability to interfere with a protein-protein interactionbetween VAP-A and the VAP-A-interacting protein, and optionally furthertesting the selected test compound for its ability to modulate cellularactivities associated with VAP-A and/or the VAP-A-interacting protein.

The present invention further provides a composition having twoexpression vectors. One vector contains a nucleic acid encoding VAP-A ora homologue, derivative or fragment thereof. Another vector contains aVAP-A-interacting protein or a homologue, derivative or fragmentthereof. In addition, an expression vector is also provided containing(1) a first nucleic acid encoding VAP-A or a homologue, derivative orfragment thereof; and (2) a second nucleic acid encoding aVAP-A-interacting protein or a homologue, derivative or fragmentthereof.

Host cells are also provided comprising the expression vector(s). Inaddition, the present invention also provides a host cell having twoexpression cassettes. One expression cassette includes a promoteroperably linked to a nucleic acid encoding VAP-A or a homologue,derivative or fragment thereof. Another expression cassette includes apromoter operably linked to a nucleic acid encoding a VAP-A-interactingprotein or a homologue, derivative or fragment thereof. Preferably, theexpression cassettes are chimeric expression cassettes with heterologouspromoters included.

In specific embodiments of the host cells or expression vectors, one ofthe two nucleic acids is linked to a nucleic acid encoding a DNA bindingdomain, and the other is linked to a nucleic acid encoding atranscription-activation domain, whereby two fusion proteins can beencoded.

In accordance with yet another aspect of the present invention, methodsare provided for modulating the functions and activities of aVAP-A-containing protein complex of the present invention, orinteracting protein members thereof. The methods may be used in treatingor preventing diseases and disorders such as diabetes, obesity,ischemia, and insulin resistance. In one embodiment, the methodcomprises reducing the protein complex concentration and/or inhibitingthe functional activities of the protein complex. Alternatively, theconcentration and/or activity of VAP-A or one of the VAP-A-interactingproteins may be reduced or inhibited. Thus, the methods may includeadministering to a patient an antibody specific to a protein complex orVAP-A or a VAP-A-interacting protein, an antisense oligo or ribozymeselectively hybridizable to a gene or mRNA encoding VAP-A or aVAP-A-interacting protein. Also useful is a compound identified in ascreening assay of the present invention capable of disrupting theinteraction between VAP-A and a VAP-A-interacting protein, or inhibitingthe activities of VAP-A and/or a VAP-A-interacting protein. In addition,gene therapy methods may also be used in reducing the expression of thegene(s) encoding VAP-A and/or a VAP-A-interacting protein.

In another embodiment, the methods for modulating the functions andactivities of a VAP-A-containing protein complex of the presentinvention or interacting protein members thereof comprises increasingthe protein complex concentration and/or activating the functionalactivities of the protein complex. Alternatively, the concentrationand/or activity of one of the VAP-A-interacting proteins or VAP-A may beincreased. Thus, a particular VAP-A-containing protein complex, VAP-A ora VAP-A-interacting protein of the present invention may be administereddirectly to a patient. Or, exogenous genes encoding one or more proteinmembers of a VAP-A-containing protein complex may be introduced into apatient by gene therapy techniques. In addition, a patient needingtreatment or prevention may also be administered with compoundsidentified in a screening assay of the present invention capable oftriggering or initiating, enhancing or stabilizing protein-proteininteractions between VAP-A or a homologue, derivative or fragmentthereof and a VAP-A-interacting protein provided in the presentinvention, or a homologue, derivative or fragment thereof.

The foregoing and other advantages and features of the invention, andthe manner in which the same are accomplished, will become more readilyapparent upon consideration of the following detailed description of theinvention taken in conjunction with the accompanying examples, whichillustrate preferred and exemplary embodiments.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

The terms “polypeptide,” “protein,” and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length of greater than two amino acids.Unless otherwise specified, the terms “polypeptide,” “protein,” and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms. Examples of modified forms include, but are not limited to,glycosylated forms, phosphorylated forms, myristoylated forms,palmitoylated forms, ribosylated forms, acetylated forms, ubiquitinatedforms, etc. Modifications also include intra-molecular crosslinking andcovalent attachment to various moieties such as lipids, flavin, biotin,polyethylene glycol or derivatives thereof, etc. In addition,modifications may also include cyclization, branching and cross-linking.Further, amino acids other than the conventional twenty amino acidsencoded by genes may also be included in a polypeptide.

As used herein, the term “interacting” or “interaction” means that twoprotein domains, fragments or complete proteins exhibit sufficientphysical affinity to each other so as to bring the two “interacting”protein domains, fragments or proteins physically close to each other.An extreme case of interaction is the formation of a chemical bond thatresults in continual and stable proximity of the two entities.Interactions that are based solely on physical affinities, althoughusually more dynamic than chemically bonded interactions, can be equallyeffective in co-localizing two proteins. Examples of physical affinitiesand chemical bonds include but are not limited to, forces caused byelectrical charge differences, hydrophobicity, hydrogen bonds, van derWaals force, ionic force, covalent linkages, and combinations thereof.The state of proximity between the interaction domains, fragments,proteins or entities may be transient or permanent, reversible orirreversible. In any event, it is in contrast to and distinguishablefrom contact caused by natural random movement of two entities.Typically, although not necessarily, an “interaction” is exhibited bythe binding between the interaction domains, fragments, proteins, orentities. Examples of interactions include specific interactions betweenantigen and antibody, ligand and receptor, enzyme and substrate, and thelike.

An “interaction” between two protein domains, fragments or completeproteins can be determined by a number of methods. For example, aninteraction can be determined by functional assays such as thetwo-hybrid systems. Protein-protein interactions can also be determinedby various biophysical and biochemical approaches based on the affinitybinding between the two interacting partners. Such biochemical methodsgenerally known in the art include, but are not limited to, proteinaffinity chromatography, affinity blotting, immunoprecipitation, and thelike. The binding constant for two interacting proteins, which reflectsthe strength or quality of the interaction, can also be determined usingmethods known in the art. See Phizicky and Fields, Microbiol. Rev.,59:94-123 (1995).

As used herein, the term “protein complex” means a composite unit thatis a combination of two or more proteins formed by interaction betweenthe proteins. Typically, but not necessarily, a “protein complex” isformed by the binding of two or more proteins together through specificnon-covalent binding interactions. However, covalent bonds may also bepresent between the interacting partners. For instance, the twointeracting partners can be covalently crosslinked so that the proteincomplex becomes more stable.

The term “protein fragment” as used herein means a polypeptide thatrepresents a portion of a protein. When a protein fragment exhibitsinteractions with another protein or protein fragment, the two entitiesare said to interact through interaction domains that are containedwithin the entities.

As used herein, the term “domain” means a functional portion, segment orregion of a protein, or polypeptide. “Interaction domain” refersspecifically to a portion, segment or region of a protein, polypeptideor protein fragment that is responsible for the physical affinity ofthat protein, protein fragment or isolated domain for another protein,protein fragment or isolated domain.

The term “isolated” when used in reference to nucleic acids (whichinclude gene sequences) of this invention is intended to mean that anucleic acid molecule is present in a form other than found in nature inits original environment with respect to its association with othermolecules. For example, since a naturally existing chromosome includes along nucleic acid sequence, an “isolated nucleic acid” as used hereinmeans a nucleic acid molecule having only a portion of the nucleic acidsequence in the chromosome but not one or more other portions present onthe same chromosome. Thus, for example, an isolated gene typicallyincludes no more than 5 kb, preferably no more than 2 kb, morepreferably no more than 1 kb naturally occurring nucleic acid sequencethat immediately flanks the gene in the naturally existing chromosome orgenomic DNA. However, it is noted that an “isolated nucleic acid” asused herein is distinct from a clone in a conventional library such asgenomic DNA library and cDNA library in that the clones in a library isstill in admixture with almost all the other nucleic acids in achromosome or a cell. An isolated nucleic acid can be in a vector. Anisolated nucleic acid can also be part of a composition so long as thecomposition is substantially different from the nucleic acid's originalnatural environment. In this respect, an isolated nucleic acid can be ina semi-purified state, i.e., in a composition having certain naturalcellular components, while it is substantially separated from othernaturally occurring nucleic acids and can be readily detected and/orassayed by standard molecular biology techniques. Preferably, an“isolated nucleic acid” is separated from at least 50%, more preferablyat least 75%, most preferably at least 90% of other naturally occurringnucleic acids.

The term “isolated nucleic acid” embraces “purified nucleic acid” whichmeans a specified nucleic acid is in a substantially homogenouspreparation of nucleic acid substantially free of other cellularcomponents, other nucleic acids, viral materials, or culture medium, orchemical precursors or by-products associated with chemical reactionsfor chemical synthesis of nucleic acids. Typically, a “purified nucleicacid” can be obtained by standard nucleic acid purification methods. Ina purified nucleic acid, preferably the specified nucleic acid moleculeconstitutes at least 75%, preferably at least 85%, and more preferablyat least 95% of the total nucleic acids in the preparation. The term“purified nucleic acid” also means nucleic acids prepared from arecombinant host cell (in which the nucleic acids have beenrecombinantly amplified and/or expressed) or chemically synthesizednucleic acids.

The term “isolated nucleic acid” also encompasses “recombinant nucleicacid” which is used herein to mean a hybrid nucleic acid produced byrecombinant DNA technology having the specified nucleic acid moleculecovalently linked to one or more nucleic acid molecules that are not thenucleic acids naturally flanking the specified nucleic acid. Typically,such one or more nucleic acid molecules flanking the specified nucleicacid are no more than 50 kb, preferably no more than 25 kb.

The term “isolated polypeptide” as used herein means a polypeptidemolecule is present in a form other than found in nature in its originalenvironment with respect to its association with other molecules.Typically, an “isolated polypeptide” is separated from at least 50%,more preferably at least 75%, most preferably at least 90% of othernaturally co-existing polypeptides in a cell or organism.

The term “isolated polypeptide” encompasses a “purified polypeptide”which is used herein to mean a specified polypeptide is in asubstantially homogenous preparation substantially free of othercellular components, other polypeptides, viral materials, or culturemedium, or when the polypeptide is chemically synthesized, chemicalprecursors or by-products associated with the chemical synthesis. For apurified polypeptide, preferably the specified polypeptide moleculeconstitutes at least 75%, preferably at least 85%, and more preferablyat least 95% of the total polypeptide in the preparation. A “purifiedpolypeptide” can be obtained from natural or recombinant host cells bystandard purification techniques, or by chemically synthesis.

The term “isolated polypeptide” also encompasses a “recombinantpolypeptide” which is used herein to mean a hybrid polypeptide producedby recombinant DNA technology or chemical synthesis having a specifiedpolypeptide molecule covalently linked to one or more polypeptidemolecules that do not naturally flank the specified polypeptide.

As used herein, the term “homologue,” when used in connection with afirst native protein or fragment thereof that is discovered, accordingto the present invention, to interact with a second native protein orfragment thereof, means a polypeptide that exhibits an amino acidsequence homology and/or structural resemblance to the first nativeinteracting protein, or to one of the interacting domains of the firstnative protein such that it is capable of interacting with the secondnative protein. Typically, a protein homologue of a native protein mayhave an amino acid sequence that is at least 50%, preferably at least75%, more preferably at least 80%, 85%, 86%, 87%, 88% or 89%, even morepreferably at least 90%, 91%, 92%, 93% or 94%, and most preferably 95%,96%, 97%, 98% or 99% identical to the native protein. Examples ofhomologues may be the ortholog proteins of other species includinganimals, plants, yeast, bacteria, and the like. Homologues may also beselected by, e.g., mutagenesis in a native protein. For example,homologues may be identified by site-specific mutagenesis in combinationwith assays for detecting protein-protein interactions, e.g., the yeasttwo-hybrid system described below, as will be apparent to skilledartisans apprised of the present invention. Other techniques fordetecting protein-protein interactions include, e.g., protein affinitychromatography, affinity blotting, in vitro binding assays, and thelike.

For the purpose of comparing two different nucleic acid or polypeptidesequences, one sequence (test sequence) may be described to be aspecific “percent identical to” another sequence (reference sequence) inthe present disclosure. In this respect, when the length of the testsequence is less than 90% of the length of the reference sequence, thepercentage identity is determined by the algorithm of Myers and Miller,Bull. Math. Biol., 51:5-37 (1989) and Myers and Miller, Comput. Appl.Biosci., 4(1):11-7 (1988). Specifically, the identity is determined bythe ALIGN program, which is available at a website maintained by IGH,Montpellier, FRANCE. The default parameters are used.

Where the length of the test sequence is at least 90% of the length ofthe reference sequence, the percentage identity is determined by thealgorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77(1993), which is incorporated into various BLAST programs. Specifically,the percentage identity is determined by the “BLAST 2 Sequences” tool,which is available at the NCBI website (National Center forBiotechnology Information). See Tatusova and Madden, FEMS Microbiol.Lett., 174(2):247-50 (1999). For pairwise DNA-DNA comparison, the BLASTN2.1.2 program is used with default parameters (Match: 1; Mismatch: −2;Open gap: 5 penalties; extension gap: 2 penalties; gap x_dropoff: 50;expect: 10; and word size: 11, with filter). For pairwiseprotein-protein sequence comparison, the BLASTP 2.1.2 program isemployed using default parameters (Matrix: BLOSUM62; gap open: 11; gapextension: 1; x_dropoff: 15; expect: 10.0; and wordsize: 3, withfilter).

The term “derivative,” when used in connection with a first nativeprotein (or fragment thereof) that is discovered, according to thepresent invention, to interact with a second native protein (or fragmentthereof), means a modified form of the first native protein prepared bymodifying the side chain groups of the first native protein withoutchanging the amino acid sequence of the first native protein. Themodified form, i.e., the derivative should be capable of interactingwith the second native protein. Examples of modified forms includeglycosylated forms, phosphorylated forms, myristylated forms,ribosylated forms, ubiquitinated forms, and the like. Derivatives alsoinclude hybrid or fusion proteins containing a native protein or afragment thereof. Methods for preparing such derivative forms should beapparent to skilled artisans. The prepared derivatives can be easilytested for their ability to interact with the native interacting partnerusing techniques known in the art, e.g., protein affinitychromatography, affinity blotting, in vitro binding assays, yeasttwo-hybrid assays, and the like.

The term “isolated protein complex” means a protein complex present in acomposition or environment that is different from that found innature—in its native or original cellular or body environment.Preferably, an “isolated protein complex” is separated from at least50%, more preferably at least 75%, most preferably at least 90% of othernaturally co-existing cellular or tissue components. Thus, an “isolatedprotein complex” may also be a naturally existing protein complex in anartificial preparation or a non-native host cell. An “isolated proteincomplex” may also be a “purified protein complex”, that is, asubstantially purified form in a substantially homogenous preparationsubstantially free of other cellular components, other polypeptides,viral materials, or culture medium, or, when the protein components inthe protein complex are chemically synthesized, free of chemicalprecursors or by-products associated with the chemical synthesis. A“purified protein complex” typically means a preparation containingpreferably at least 75%, more preferably at least 85%, and mostpreferably at least 95% a particular protein complex. A “purifiedprotein complex” may be obtained from natural or recombinant host cellsor other body samples by standard purification techniques, or bychemical synthesis.

The terms “hybrid protein,” “hybrid polypeptide,” “hybrid peptide,”“fusion protein,” “fusion polypeptide,” and “fusion peptide” are usedherein interchangeably to mean a non-naturally occurring protein havinga specified polypeptide molecule covalently linked to one or morepolypeptide molecules that do not naturally link to the specifiedpolypeptide. Thus, a “hybrid protein” may be two naturally occurringproteins or fragments thereof linked together by a covalent linkage. A“hybrid protein” may also be a protein formed by covalently linking twoartificial polypeptides together. Typically but not necessarily, the twoor more polypeptide molecules are linked or “fused” together by apeptide bond forming a single non-branched polypeptide chain.

The term “antibody” as used herein encompasses both monoclonal andpolyclonal antibodies that fall within any antibody classes, e.g., IgG,IgM, IgA, or derivatives thereof. The term “antibody” also includesantibody fragments including, but not limited to, Fab, F(ab′)₂, andconjugates of such fragments, and single-chain antibodies comprising anantigen recognition epitope. In addition, the term “antibody” also meanshumanized antibodies, including partially or fully humanized antibodies.An antibody may be obtained from an animal, or from a hybridoma cellline producing a monoclonal antibody, or obtained from cells orlibraries recombinantly expressing a gene encoding a particularantibody.

The term “selectively immunoreactive” as used herein means that anantibody is reactive thus binds to a specific protein or proteincomplex, but not other similar proteins or fragments or componentsthereof.

The term “activity” when used in connection with proteins or proteincomplexes means any physiological or biochemical activities displayed byor associated with a particular protein or protein complex including butnot limited to activities exhibited in biological processes and cellularfunctions, ability to interact with or bind another molecule or a moietythereof, binding affinity or specificity to certain molecules, in vitroor in vivo stability (e.g., protein degradation rate, or in the case ofprotein complexes ability to maintain the form of protein complex),antigenicity and immunogenecity, enzymatic activities, etc. Suchactivities may be detected or assayed by any of a variety of suitablemethods as will be apparent to skilled artisans.

The term “compound” as used herein encompasses all types of organic orinorganic molecules, including but not limited proteins, peptides,polysaccharides, lipids, nucleic acids, small organic molecules,inorganic compounds, and derivatives thereof.

As used herein, the term “interaction antagonist” means a compound thatinterferes with, blocks, disrupts or destabilizes a protein-proteininteraction; blocks or interferes with the formation of a proteincomplex; or destabilizes, disrupts or dissociates an existing proteincomplex.

The term “interaction agonist” as used herein means a compound thattriggers, initiates, propagates, nucleates, or otherwise enhances theformation of a protein-protein interaction; triggers, initiates,propagates, nucleates, or otherwise enhances the formation of a proteincomplex; or stabilizes an existing protein complex.

Unless otherwise specified, the term “VAP-A” as used herein means thehuman VAP-A protein. The usage for naming other proteins should besimilar unless otherwise specified in the present disclosure.

2. Protein Complexes

Novel protein-protein interactions have been discovered and confirmedusing yeast two-hybrid systems. In particular, it has been discoveredthat VAP-A specifically interacts with proteins including PPP1R3 andGTR3. Specific fragments capable of conferring interacting properties onVAP-A, and the VAP-A-interacting proteins have also been identified,which are summarized in Table 1. The GenBank Reference Numbers for thecDNA sequences encoding VAP-A, and the VAP-A-interacting proteins arenoted in Table 1 below.

TABLE 1 Binding Regions or VAP-A and Its Interacting Partners BaitProtein Prey Proteins Name and Amino Acid GenBank Amino Acid GenBankCoordinates Accession Coordinates Accession No. Start Stop Names Nos.Start Stop VAP-A 90 243 PPP1R3 NM_002711 686 1122 (GenBank GTR3NM_006931 358 494 Accession No. GTR3 NM_006931 358 496 NM_003574)

2.1. Biological Significance

We have demonstrated an interaction between VAP-A and the glucosetransporter-like protein III (GTR3) in a yeast two-hybrid search of abrain library. We have also demonstrated an interaction between VAP-Aand protein phosphatase 1, regulatory subunit 3 (PPP1R3) in a yeasttwo-hybrid search of a skeletal muscle library. Northern blot analysisshows that VAP-A is expressed in all human tissues tested. In mousetissues, VAP-A is most highly expressed in brain, skeletal muscle,kidney, testis, and ovary. VAP-A was identified in Aplysia as a VAMP(vesicle-associated membrane protein) associated protein. This bindingappears to be specific as VAP-A does not bind to SNAP-25. VAMP, alongwith other SNARE proteins, is involved in the docking and fusion ofmembrane vesicles to target organelle membranes. Like VAMP, VAP-Acontains a C-terminal transmembrane region that tethers the proteins tothe membrane. Due to its association with VAMP and SNAP-25, it is likelyto be involved in the docking and fusion steps of insulin-stimulatedGLUT4 translocation.

Glucose transporter-like protein 3 (GTR3) is involved in cellular uptakeof glucose. It is expressed primarily in the brain, although it is foundin other tissues such as skeletal muscle. Like the insulin-responsiveglucose transporter, GLUT4, GTR3 is also been shown to redistribute tothe plasma membrane following insulin treatment. However, the majorityof GTR3 is found on the cell surface, although a small fraction is foundin secretory vesicles that also contain SNARE proteins such as VAMP(Thoidis et al., J. Biol. Chem. 274:14062, 1999). It is unclear whetherregulation of GTR3 in the brain involved compartmentalization, as is thecase with GLUT4 in adipose and skeletal muscle. It does appear that GTR3is regulated by a different mechanism than is GLUT4, since prolongedenergy demands on cultured skeletal muscle cells cause an upregulationof GTR3 protein levels (Khayat et al., Biochem. J. 333:713, 1998).Likewise, Reagan et al. (Am. Physiol. Soc. E879, 1999) demonstrated thatGTR3 protein levels are increased in the hippocampus under diabeticconditions. Maintenance of glucose regulation is highly importance, aschronic stress and resulting glucocorticoid levels can renderhippocampal neurons vulnerable to neurotoxic events by decreasingneuronal glucose utilization (McEwen and Sapolsky, Curr. Opin.Neurobiol. 5:205, 1995). Thus, understanding how GTR3 expression,activity, and cellular localization is regulated may be important toincreasing neuronal survival following ischemia and other neurotoxicevents. The interaction between VAP-A and Glut3 may provide a mechanismfor neurons to regulate their glucose intake by GTR3. Additionally,VAP-A appears to be involved in fusion of Glut4-containing vesicles withthe plasma membrane of insulin-responsive cells, thus suggesting thatVAP-A plays a role in insulin resistance (Foster and Klip, Am. J.Physiol. Cell. Physiol. 279(4):C877-90 2000).

Protein phosphatase 1, regulatory subunit 3 (PPP1R3) is a glycogen andsarcoplasmic reticulum-binding serine/threonine phosphatase. It haslimited expression in lung, skeletal muscle, and testis. Proteinphosphatase 1 functions as a heterodimer consisting of a 37-kD catalyticsubunit and a 124-kD regulatory subunit. PPP1R3 binds to muscle glycogenand enhances dephosphorylation of glycogen-bound substrates such asglycogen synthase and glycogen phosphorylase kinase. Phosphorylation atser46 of the PPP3R1 subunit in response to insulin increases proteinphosphatase 1 activity, while phosphorylation at ser65 in response toadrenaline causes dissociation of the catalytic subunit from the Gsubunit and inhibits glycogen synthesis. PPP3R1 is thus likely to beinvolved in type II diabetes and/or obesity. A polymorphism in the3′-untranslated region of PPP3R1 is associated with insulin resistancein Pima Indians (Xia et al., Diabetes 47:1519, 1998). Additionally, acommon D905Y polymorphism was identified that was associated withinsulin resistance (Hansen et al., Hum. Molec. Genet. 4:1313, 1995).These authors propose that this variant and obesity factors interact toinduce hypersecretion of insulin. Although mutations in PPP3R1 only maynot manifest as a full-blown insulin resistant phenotype, this may occurin conjunction with other genetic or biochemical problems. Theinteraction between VAP-A and PPP3R1 may represent a link between aprotein with a genetic link to insulin resistance, and a protein with abiochemical link to insulin action.

The interactions between VAP-A and the VAP-A-interacting proteinssuggest that these proteins are involved in common biological processesincluding, but not limited to, docking and fusion of membrane vesiclesto target organelle membranes and cellular uptake of glucose, anddisease pathways involving such cellular functions.

2.2. Protein Complexes

Accordingly, the present invention provides protein complexes formedbetween VAP-A and one or more VAP-A-interacting proteins selected fromthe group consisting of PPP1R3 and GTR3. The present invention alsoprovides protein complexes formed from the interaction betweenhomologues, derivatives or fragments of VAP-A and one or more of theVAP-A-interacting proteins in accordance with the present invention. Inaddition, the present invention further encompasses protein complexeshaving VAP-A and homologues, derivatives or fragments of one or more ofthe VAP-A-interacting proteins in accordance with the present invention.In yet another embodiment, protein complexes are provided havinghomologues, derivatives or fragments of VAP-A and homologues,derivatives or fragments of one or more of the VAP-A-interactingproteins in accordance with the present invention. In other words, oneor more of the interacting protein members of a protein complex of thepresent invention may be a native protein or a homologue, derivative orfragment of a native protein.

Thus, for example, one interacting partner in a protein complex can be acomplete native VAP-A, a VAP-A homologue capable of interacting with,e.g., PPP1R3, a VAP-A derivative, a derivative of the VAP-A homologue, aVAP-A fragment capable of interacting with PPP1R3 (VAP-A fragment(s)containing the coordinates shown in Table 1), a derivative of the VAP-Afragment, or a fusion protein containing (1) complete native VAP-A, (2)a VAP-A homologue capable of interacting with PPP1R3 or (3) a VAP-Afragment capable of interacting with PPP1R3. Besides native PPP1R3,useful interacting partners for VAP-A or a homologue or derivative orfragment thereof also include homologues of PPP1R3 capable ofinteracting with VAP-A, derivatives of the native or homologue PPP1R3capable of interacting with VAP-A, fragments of the PPP1R3 capable ofinteracting with VAP-A (e.g., a fragment containing the identifiedinteracting regions shown in Table 1), derivatives of the PPP1R3fragments, or fusion proteins containing (1) a complete PPP1R3, (2) aPPP1R3 homologue capable of interacting with VAP-A or (3) a PPP1R3fragment capable of interacting with VAP-A.

PPP1R3 fragments capable of interacting with VAP-A can be identified bythe combination of molecular engineering of a PPP1R3-encoding nucleicacid and a method for testing protein-protein interaction. For example,the coordinates in Table 1 can be used as starting points and variousPPP1R3 fragments falling within the coordinates can be generated bydeletions from either or both ends of the coordinates. The resultingfragments can be tested for their ability to interact with VAP-A usingany methods known in the art for detecting protein-protein interactions(e.g., yeast two-hybrid method). Alternatively, various PPP1R3 fragmentscan be made by chemical synthesis. The PPP1R3 fragments can then betested for its ability to interact with VAP-A using any method known inthe art for detecting protein-protein interactions. Examples of suchmethods include protein affinity chromatography, affinity blotting, invitro binding assays, yeast two-hybrid assays, and the like. Likewise,VAP-A fragments capable of interacting with PPP1R3 can also beidentified in a similar manner.

Other protein complexes can be formed in a similar manner based oninteractions between VAP-A and its other interacting partners discoveredaccording to the present invention or homologues, derivatives orfragments of such other interacting partners. In addition, proteincomplexes containing VAP-A and two or more members of the group ofPPP1R3 and GTR3 or homologues, derivatives, or fragments thereof canalso be formed.

In a specific embodiment of the protein complex of the presentinvention, two or more interacting partners (VAP-A and one or moreproteins selected from the group consisting of PPP1R3 and GTR3, orhomologues, derivatives or fragments thereof) are directly fusedtogether, or covalently linked together through a peptide linker,forming a hybrid protein having a single unbranched polypeptide chain.Thus, the protein complex may be formed by “intramolecular” interactionsbetween two portions of the hybrid protein. Again, one or both of thefused or linked interacting partners in this protein complex may be anative protein or a homologue, derivative or fragment of a nativeprotein.

The protein complexes of the present invention can also be in a modifiedform. For example, an antibody selectively immunoreactive with theprotein complex can be bound to the protein complex. In another example,a non-antibody modulator capable of enhancing the interaction betweenthe interacting partners in the protein complex may be included.Alternatively, the protein members in the protein complex may becross-linked for purposes of stabilization. Various crosslinking methodsmay be used. For example, a bifunctional reagent in the form of R-S-S-R′may be used in which the R and R′ groups can react with certain aminoacid side chains in the protein complex forming covalent linkages. Seee.g., Traut et al., in Creighton ed., Protein Function: A PracticalApproach, IRL Press, Oxford, 1989; Baird et al., J. Biol. Chem.,251:6953-6962 (1976). Other useful crosslinking agents include, e.g.,Denny-Jaffee reagent, a heterbiofunctional photoactivable moietycleavable through an azo linkage (See Denny et al., Proc. Natl. Acad.Sci. USA, 81:5286-5290 (1984)), and¹²⁵I-{S-[N-(3-iodo-4-azidosalicyl)cysteaminyl]-2-thiopyridine}, acysteine-specific photocrosslinking reagent (see Chen et al., Science,265:90-92 (1994)).

The above-described protein complexes may further include any additionalcomponents, e.g., other proteins, nucleic acids, lipid molecules,monosaccharides or polysaccharides, ions, etc.

2.3. Methods of Preparing Protein Complexes

The protein complex of the present invention can be prepared by avariety of methods. Specifically, a protein complex can be isolateddirectly from an animal tissue sample, preferably a human tissue samplecontaining the protein complex. Alternatively, a protein complex can bepurified from host cells that recombinantly express the members of theprotein complex. As will be apparent to a skilled artisan, a proteincomplex can be prepared from a tissue sample or recombinant host cellsby coimmunoprecipitation using an antibody immunoreactive with aninteracting protein partner, or preferably an antibody selectivelyimmunoreactive with the protein complex as will be discussed in detailbelow.

The antibodies can be monoclonal or polyclonal. Coimmunoprecipitation isa commonly used method in the art for isolating or detecting boundproteins. In this procedure, generally a serum sample or tissue or celllysate is admixed with a suitable antibody. The protein complex bound tothe antibody is precipitated and washed. The bound protein complexes arethen eluted.

Alternatively, immunoaffinity chromatography and immunoblotingtechniques may also be used in isolating the protein complexes fromnative tissue samples or recombinant host cells using an antibodyimmunoreactive with an interacting protein partner, or preferably anantibody selectively immunoreactive with the protein complex. Forexample, in protein immunoaffinity chromatography, the antibody iscovalently or non-covalently coupled to a matrix (e.g., Sepharose),which is then packed into a column. Extract from a tissue sample, orlysate from recombinant cells is passed through the column where itcontacts the antibodies attached to the matrix. The column is thenwashed with a low-salt solution to wash away the unbound or loosely(non-specifically) bound components. The protein complexes that areretained in the column can be then eluted from the column using ahigh-salt solution, a competitive antigen of the antibody, a chaotropicsolvent, or sodium dodecyl sulfate (SDS), or the like. Inimmunoblotting, crude proteins samples from a tissue sample extract orrecombinant host cell lysate are fractionated by polyacrylamide gelelectrophoresis (PAGE) and then transferred to a membrane, e.g.,nitrocellulose. Components of the protein complex can then be located onthe membrane and identified by a variety of techniques, e.g., probingwith specific antibodies.

In another embodiment, individual interacting protein partners may beisolated or purified independently from tissue samples or recombinanthost cells using similar methods as described above. The individualinteracting protein partners are then combined under conditionsconducive to their interaction thereby forming a protein complex of thepresent invention. It is noted that different protein-proteininteractions may require different conditions. As a starting point, forexample, a buffer having 20 mM Tris-HCl, pH 7.0 and 500 mM NaCl may beused. Several different parameters may be varied, including temperature,pH, salt concentration, reducing agent, and the like. Some minor degreeof experimentation may be required to determine the optimum incubationcondition, this being well within the capability of one skilled in theart once apprised of the present disclosure.

In yet another embodiment, the protein complex of the present inventionmay be prepared from tissue samples or recombinant host cells or othersuitable sources by protein affinity chromatography or affinityblotting. That is, one of the interacting protein partners is used toisolate the other interacting protein partner(s) by binding affinitythus forming protein complexes. Thus, an interacting protein partnerprepared by purification from tissue samples or by recombinantexpression or chemical synthesis may be bound covalently ornon-covalently to a matrix, e.g., Sepharose, which is then packed into achromatography column. The tissue sample extract or cell lysate from therecombinant cells can then be contacted with the bound protein on thematrix. A low-salt solution is used to wash off the unbound or looselybound components, and a high-salt solution is then employed to elute thebound protein complexes in the column. In affinity blotting, crudeprotein samples from a tissue sample or recombinant host cell lysate canbe fractionated by polyacrylamide gel electrophoresis (PAGE) and thentransferred to a membrane, e.g., nitrocellulose. The purifiedinteracting protein member is then bound to its interacting proteinpartner(s) on the membrane forming protein complexes, which are thenisolated from the membrane.

It will be apparent to skilled artisans that any recombinant expressionmethods may be used in the present invention for purposes of expressingthe protein complexes or individual interacting proteins. Generally, anucleic acid encoding an interacting protein member can be introducedinto a suitable host cell. For purposes of forming a recombinant proteincomplex within a host cell, nucleic acids encoding two or moreinteracting protein members should be introduced into the host cell.

Typically, the nucleic acids, preferably in the form of DNA, areincorporated into a vector to form expression vectors capable ofdirecting the production of the interacting protein member(s) onceintroduced into a host cell. Many types of vectors can be used for thepresent invention. Methods for the construction of an expression vectorfor purposes of this invention should be apparent to skilled artisansapprised of the present disclosure. See generally, Current Protocols inMolecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. &Wiley lnterscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRLPress, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods inEnzymology 153:516-544 (1987); The Molecular Biology of the YeastSaccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. Iand II, 1982; and Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, 1989.

Generally, the expression vectors include an expression cassette havinga promoter operably linked to a DNA encoding an interacting proteinmember. The promoter can be a native promoter, i.e., the promoter foundin naturally occurring cells to be responsible for the expression of theinteracting protein member in the cells. Alternatively, the expressioncassette can be a chimeric one, i.e., having a heterologous promoterthat is not the native promoter responsible for the expression of theinteracting protein member in naturally occurring cells. The expressionvector may further include an origin of DNA replication for thereplication of the vectors in host cells. Preferably, the expressionvectors also include a replication origin for the amplification of thevectors in, e.g., E. coli, and selection marker(s) for selecting andmaintaining only those host cells harboring the expression vectors.Additionally, the expression cassettes preferably also contain inducibleelements, which function to control the transcription from the DNAencoding an interacting protein member. Other regulatory sequences suchas transcriptional enhancer sequences and translation regulationsequences (e.g., Shine-Dalgarno sequence) can also be operably includedin the expression cassettes. Termination sequences such as thepolyadenylation signals from bovine growth hormone, SV40, lacZ andAcMNPV polyhedral protein genes may also be operably linked to the DNAencoding an interacting protein member in the expression cassettes. Anepitope tag coding sequence for detection and/or purification of theexpressed protein can also be operably linked to the DNA encoding aninteracting protein member such that a fusion protein is expressed.Examples of useful epitope tags include, but are not limited to,influenza virus hemagglutinin (HA), Simian Virus 5 (V5), polyhistidine(6xHis), c-myc, lacZ, GST, and the like. Proteins with polyhistidinetags can be easily detected and/or purified with Ni affinity columns,while specific antibodies immunoreactive with many epitope tags aregenerally commercially available. The expression vectors may alsocontain components that direct the expressed protein extracellularly orto a particular intracellular compartment. Signal peptides, nuclearlocalization sequences, endoplasmic reticulum retention signals,mitochondrial localization sequences, myristoylation signals,palmitoylation signals, and transmembrane sequences are example ofoptional vector components that can determine the destination ofexpressed proteins. When it is desirable to express two or moreinteracting protein members in a single host cell, the DNA fragmentsencoding the interacting protein members may be incorporated into asingle vector or different vectors.

The thus constructed expression vectors can be introduced into the hostcells by any techniques known in the art, e.g., by direct DNAtransformation, microinjection, electroporation, viral infection,lipofection, gene gun, and the like. The expression of the interactingprotein members may be transient or stable. The expression vectors canbe maintained in host cells in an extrachromosomal state, i.e., asself-replicating plasmids or viruses. Alternatively, the expressionvectors can be integrated into chromosomes of the host cells byconventional techniques such as selection of stable cell lines orsite-specific recombination. In stable cell lines, at least theexpression cassette portion of the expression vector is integrated intoa chromosome of the host cells.

The vector construct can be designed to be suitable for expression invarious host cells, including but not limited to bacteria, yeast cells,plant cells, insect cells, and mammalian and human cells. Methods forpreparing expression vectors for expression in different host cellsshould be apparent to a skilled artisan.

Homologues and fragments of the native interacting protein members canalso be easily expressed using the recombinant methods described above.For example, to express a protein fragment, the DNA fragmentincorporated into the expression vector can be selected such that itonly encodes the protein fragment. Likewise, a specific hybrid proteincan be expressed using a recombinant DNA encoding the hybrid protein.Similarly, a homologue protein may be expressed from a DNA sequenceencoding the homologue protein. A homologue-encoding DNA sequence may beobtained by manipulating the native protein-encoding sequence usingrecombinant DNA techniques. For this purpose, random or site-directedmutagenesis can be conducted using techniques generally known in theart. To make protein derivatives, for example, the amino acid sequenceof a native interacting protein member may be changed in predeterminedmanners by site-directed DNA mutagenesis to create or remove consensussequences for, e.g., phosphorylation by protein kinases, glycosylation,ribosylation, myristolation, palmytoylation, ubiquitination, and thelike. Alternatively, non-natural amino acids can be incorporated into aninteracting protein member during the synthesis of the protein inrecombinant host cells. For example, photoreactive lysine derivativescan be incorporated into an interacting protein member duringtranslation by using a modified lysyl-tRNA. See, e.g., Wiedmann et al.,Nature, 328:830-833 (1989); Musch et al., Cell, 69:343-352 (1992). Otherphotoreactive amino acid derivatives can also be incorporated in asimilar manner. See, e.g., High et al., J. Biol. Chem., 368:28745-28751(1993). Indeed, the photoreactive amino acid derivatives thusincorporated into an interacting protein member can function tocross-link the protein to its interacting protein partner in a proteincomplex under predetermined conditions.

In addition, derivatives of the native interacting protein members ofthe present invention can also be prepared by chemically linking certainmoieties to amino acid side chains of the native proteins.

If desired, the homologues and derivatives thus generated can be testedto determine whether they are capable of interacting with their intendedpartners to form protein complexes. Testing can be conducted by e.g.,the yeast two-hybrid system or other methods known in the art fordetecting protein-protein interaction.

A hybrid protein as described above having VAP-A or a homologue,derivative, or fragment thereof covalently linked by a peptide bond or apeptide linker to a protein selected from the group consisting of PPP1R3and GTR3 or a homologue, derivative, or fragment thereof, can beexpressed recombinantly from a chimeric nucleic acid, e.g., a DNA ormRNA fragment encoding the fusion protein. Accordingly, the presentinvention also provides a nucleic acid encoding the hybrid protein ofthe present invention. In addition, an expression vector havingincorporated therein a nucleic acid encoding the hybrid protein of thepresent invention is also provided. The methods for making such chimericnucleic acids and expression vectors containing them should be apparentto skilled artisans apprised of the present disclosure.

2.4. Protein Microchip

In accordance with another embodiment of the present invention, aprotein microchip or microarray is provided having one or more of theprotein complexes and/or antibodies selectively immunoreactive with theprotein complexes of the present invention. Protein microarrays arebecoming increasingly important in both proteomics research andprotein-based detection and diagnosis of diseases. The proteinmicroarrays in accordance with this embodiment of the present inventionwill be useful in a variety of applications including, e.g., large-scaleor high-throughput screening for compounds capable of binding to theprotein complexes or modulating the interactions between the interactingprotein members in the protein complexes.

The protein microarray of the present invention can be prepared in anumber of methods known in the art. An example of a suitable method isthat disclosed in MacBeath and Schreiber, Science, 289:1760-1763 (2000).Essentially, glass microscope slides are treated with analdehyde-containing silane reagent (SuperAldehyde Substrates purchasedfrom TeleChem International, Cupertino, Calif.). Nanoliter volumes ofprotein samples in a phosphate-buffered saline with 40% glycerol arethen spotted onto the treated slides using a high-precisioncontact-printing robot. After incubation, the slides are immersed in abovine serum albumin (BSA)-containing buffer to quench the unreactedaldehydes and to form a BSA layer that functions to prevent non-specificprotein binding in subsequent applications of the microchip.Alternatively, as disclosed in MacBeath and Schreiber, proteins orprotein complexes of the present invention can be attached to a BSA-NHSslide by covalent linkages. BSA-NHS slides are fabricated by firstattaching a molecular layer of BSA to the surface of glass slides andthen activating the BSA with N,N′-disuccinimidyl carbonate. As a result,the amino groups of the lysine, aspartate, and glutamate residues on theBSA are activated and can form covalent urea or amide linkages withprotein samples spotted on the slides. See MacBeath and Schreiber,Science, 289:1760-1763 (2000).

Another example of a useful method for preparing the protein microchipof the present invention is that disclosed in PCT Publication Nos. WO00/4389A2 and WO 00/04382, both of which are assigned to Zyomyx and areincorporated herein by reference. First, a substrate or chip base iscovered with one or more layers of thin organic film to eliminate anysurface defects, insulate proteins from the base materials, and toensure uniform protein array. Next, a plurality of protein-capturingagents (e.g., antibodies, peptides, etc.) are arrayed and attached tothe base that is covered with the thin film. Proteins or proteincomplexes can then be bound to the capturing agents forming a proteinmicroarray. The protein microchips are kept in flow chambers with anaqueous solution.

The protein microarray of the present invention can also be made by themethod disclosed in PCT Publication No. WO 99/36576 assigned to PackardBioscience Company, which is incorporated herein by reference. Forexample, a three-dimensional hydrophilic polymer matrix, i.e., a gel, isfirst dispensed on a solid substrate such as a glass slide. The polymermatrix gel is capable of expanding or contracting and contains acoupling reagent that reacts with amine groups. Thus, proteins andprotein complexes can be contacted with the matrix gel in an expandedaqueous and porous state to allow reactions between the amine groups onthe protein or protein complexes with the coupling reagents thusimmobilizing the proteins and protein complexes on the substrate.Thereafter, the gel is contracted to embed the attached proteins andprotein complexes in the matrix gel.

Alternatively, the proteins and protein complexes of the presentinvention can be incorporated into a commercially available proteinmicrochip, e.g., the ProteinChip System from Ciphergen Biosystems Inc.,Palo Alto, Calif. The ProteinChip System comprises metal chips having atreated surface, which interact with proteins. Basically, a metal chipsurface is coated with a silicon dioxide film. The molecules of interestsuch as proteins and protein complexes can then be attached covalentlyto the chip surface via a silane coupling agent.

The protein microchips of the present invention can also be preparedwith other methods known in the art, e.g., those disclosed in U.S. Pat.Nos. 6,087,102, 6,139,831, 6,087,103; PCT Publication Nos. WO 99/60156,WO 99/39210, WO 00/54046, WO 00/53625, WO 99/51773, WO 99/35289, WO97/42507, WO 01/01142, WO 00/63694, WO 00/61806, WO 99/61148, WO99/40434, all of which are incorporated herein by reference.

3. Antibodies

In accordance with another aspect of the present invention, an antibodyimmunoreactive against a protein complex of the present invention isprovided. In one embodiment, the antibody is selectively immunoreactivewith a protein complex of the present invention. Specifically, thephrase “selectively immunoreactive with a protein complex” as usedherein means that the immunoreactivity of the antibody of the presentinvention with the protein complex is substantially higher than thatwith the individual interacting members of the protein complex so thatthe binding of the antibody to the protein complex is readilydistinguishable from the binding of the antibody to the individualinteracting member proteins based on the strength of the bindingaffinities. Preferably, the binding constants differ by a magnitude ofat least 2 fold, more preferably at least 5 fold, even more preferablyat least 10 fold, and most preferably at least 100 fold. In a specificembodiment, the antibody is not substantially immunoreactive with theinteracting protein members of the protein complex.

The antibody of the present invention can be readily prepared usingprocedures generally known in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.Typically, the protein complex against which the antibody to begenerated will be immunoreactive is used as the antigen for the purposeof producing immune response in a host animal. In one embodiment, theprotein complex used consists the native proteins. Preferably, theprotein complex includes only the interaction domain(s) of VAP-A and theinteraction domain(s) of one or more proteins selected from the groupconsisting of PPP1R3 and GTR3. As a result, a greater portion of thetotal antibodies may be selectively immunoreactive with the proteincomplexes. The interaction domains can be selected from, e.g., thoseregions summarized in Table 1. In addition, various techniques known inthe art for predicting epitopes may also be employed to design antigenicpeptides based on the interacting protein members in a protein complexof the present invention to increase the possibility of producing anantibody selectively immunoreactive with the protein complex. Suitableepitope-prediction computer programs include, e.g., MacVector fromInternational Biotechnologies, Inc. and Protean from DNAStar.

In a specific embodiment, a hybrid protein as described above in Section2.1 is used as an antigen which has VAP-A or a homologue, derivative, orfragment thereof covalently linked by a peptide bond or a peptide linkerto a protein selected from the group consisting of PPP1R3 and GTR3 or ahomologue, derivative, or fragment thereof. In a preferred embodiment,the hybrid protein consists of two interacting domains selected from theregions identified in Table 1, or homologues or derivatives thereof,covalently linked together by a peptide bond or a linker molecule.

The antibody of the present invention can be a polyclonal antibody to aprotein complex of the present invention. To produce the polyclonalantibody, various animal hosts can be employed, including, e.g., mice,rats, rabbits, goats, guinea pigs, hamsters, etc. A suitable antigenwhich is a protein complex of the present invention or a derivativethereof as described above can be administered directly to a host animalto illicit immune reactions. Alternatively, it can be administeredtogether with a carrier such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA), ovalbumin, and Tetanus toxoid. Optionally, theantigen is conjugated to a carrier by a coupling agent such ascarbodiimide, glutaraldehyde, and MBS. Any conventional adjuvants may beused to boost the immune response of the host animal to the proteincomplex antigen. Suitable adjuvants known in the art include but are notlimited to Complete Freund's Adjuvant (which contains killedmycobacterial cells and mineral oil), incomplete Freund's Adjuvant(which lacks the cellular components), aluminum salts, MF59 fromBiocine, monophospholipid, synthetic trehalose dicorynomycolate (TDM)and cell wall skeleton (CWS) both from Corixa Corp., Seattle, Wash.,non-ionic surfactant vesicles (NISV) from Proteus International PLC,Cheshire, U.K., and saponins. The antigen preparation can beadministered to a host animal by subcutaneous, intramuscular,intravenous, intradermal, or intraperitoneal injection, or by injectioninto a lymphoid organ.

The antibodies of the present invention may also be monoclonal. Suchmonoclonal antibodies may be developed using any conventional techniquesknown in the art. For example, the popular hybridoma method disclosed inKohler and Milstein, Nature, 256:495-497 (1975) is now a well-developedtechnique that can be used in the present invention. See U.S. Pat. No.4,376,110, which is incorporated herein by reference. Essentially,B-lymphocytes producing a polyclonal antibody against a protein complexof the present invention can be fused with myeloma cells to generate alibrary of hybridoma clones. The hybridoma population is then screenedfor antigen binding specificity and also for immunoglobulin class(isotype). In this manner, pure hybridoma clones producing specifichomogenous antibodies can be selected. See generally, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.Alternatively, other techniques known in the art may also be used toprepare monoclonal antibodies, which include but are not limited to theEBV hybridoma technique, the human N-cell hybridoma technique, and thetrioma technique.

In addition, antibodies selectively immunoreactive with a proteincomplex of the present invention may also be recombinantly produced. Forexample, cDNAs prepared by PCR amplification from activatedB-lymphocytes or hybridomas may be cloned into an expression vector toform a cDNA library, which is then introduced into a host cell forrecombinant expression. The cDNA encoding a specific desired protein maythen be isolated from the library. The isolated cDNA can be introducedinto a suitable host cell for the expression of the protein. Thus,recombinant techniques can be used to produce specific nativeantibodies, hybrid antibodies capable of simultaneous reaction with morethan one antigen, chimeric antibodies (e.g., the constant and variableregions are derived from different sources), univalent antibodies thatcomprise one heavy and light chain pair coupled with the Fc region of athird (heavy) chain, Fab proteins, and the like. See U.S. Pat. No.4,816,567; European Patent Publication No. 0088994; Munro, Nature,312:597 (1984); Morrison, Science, 229:1202 (1985); Oi et al.,BioTechniques, 4:214 (1986); and Wood et al., Nature, 314:446-449(1985), all of which are incorporated herein by reference. Antibodyfragments such as Fv fragments, single-chain Fv fragments (scFv), Fab′fragments, and F(ab′)₂ fragments can also be recombinantly produced bymethods disclosed in, e.g., U.S. Pat. No. 4,946,778; Skerra & Plückthun,Science, 240:1038-1041(1988); Better et al., Science, 240:1041-1043(1988); and Bird, et al., Science, 242:423-426 (1988), all of which areincorporated herein by reference.

In a preferred embodiment, the antibodies provided in accordance withthe present invention are partially or fully humanized antibodies. Forthis purpose, any methods known in the art may be used. For example,partially humanized chimeric antibodies having V regions derived fromthe tumor-specific mouse monoclonal antibody, but human C regions aredisclosed in Morrison and Oi, Adv. Immunol., 44:65-92 (1989). Inaddition, fully humanized antibodies can be made using transgenicnon-human animals. For example, transgenic non-human animals such astransgenic mice can be produced in which endogenous immunoglobulin genesare suppressed or deleted, while heterologous antibodies are encodedentirely by exogenous immunoglobulin genes, preferably humanimmunoglobulin genes, recombinantly introduced into the genome. Seee.g., U.S. Pat. Nos. 5,530,101; 5,545,806; 6,075,181; PCT PublicationNo. WO 94/02602; Green et. al., Nat. Genetics, 7: 13-21 (1994); andLonberg et al., Nature 368: 856-859 (1994), all of which areincorporated herein by reference. The transgenic non-human host animalmay be immunized with suitable antigens such as a protein complex of thepresent invention or one or more of the interacting protein membersthereof to illicit specific immune response thus producing humanizedantibodies. In addition, cell lines producing specific humanizedantibodies can also be derived from the immunized transgenic non-humananimals. For example, mature B-lymphocytes obtained from a transgenicanimal producing humanized antibodies can be fused to myeloma cells andthe resulting hybridoma clones may be selected for specific humanizedantibodies with desired binding specificities. Alternatively, cDNAs maybe extracted from mature B-lymphocytes and used in establishing alibrary that is subsequently screened for clones encoding humanizedantibodies with desired binding specificities.

In yet another embodiment, a bifunctional antibody is provided that hastwo different antigen binding sites, each being specific to a differentinteracting protein member in a protein complex of the presentinvention. The bifunctional antibody may be produced using a variety ofmethods known in the art. For example, two different monoclonalantibody-producing hybridomas can be fused together. One of the twohybridomas may produce a monoclonal antibody specific against aninteracting protein member of a protein complex of the presentinvention, while the other hybridoma generates a monoclonal antibodyimmunoreactive with another interacting protein member of the proteincomplex. The thus formed new hybridoma produces different antibodiesincluding a desired bifunctional antibody, i.e., an antibodyimmunoreactive with both of the interacting protein members. Thebifunctional antibody can be readily purified. See Milstein and Cuello,Nature, 305:537-540 (1983).

Alternatively, a bifunctional antibody may also be produced usingheterobifunctional crosslinkers to chemically link two differentmonoclonal antibodies, each being immunoreactive with a differentinteracting protein member of a protein complex. Therefore, theaggregate will bind to two interacting protein members of the proteincomplex. See Staerz et al, Nature, 314:628-631(1985); Perez et al,Nature, 316:354-356 (1985).

In addition, bifunctional antibodies can also be produced byrecombinantly expressing light and heavy chain genes in a hybridoma thatitself produces a monoclonal antibody. As a result, a mixture ofantibodies including a bifunctional antibody is produced. See DeMonte etal, Proc. Natl. Acad. Sci., USA, 87:2941-2945 (1990); Lenz and Weidle,Gene, 87:213-218 (1990).

Preferably, a bifunctional antibody in accordance with the presentinvention is produced by the method disclosed in U.S. Pat. No.5,582,996, which is incorporated herein by reference. For example, twodifferent Fabs can be provided and mixed together. The first Fab canbind to an interacting protein member of a protein complex, and has aheavy chain constant region having a first complementary domain notnaturally present in the Fab but capable of binding a secondcomplementary domain. The second Fab is capable of binding anotherinteracting protein member of the protein complex, and has a heavy chainconstant region comprising a second complementary domain not naturallypresent in the Fab but capable of binding to the first complementarydomain. Each of the two complementary domains is capable of stablybinding to the other but not to itself. For example, the leucine zipperregions of c-fos and c-jun oncogenes may be used as the first and secondcomplementary domains. As a result, the first and second complementarydomains interact with each other to form a leucine zipper thusassociating the two different Fabs into a single antibody constructcapable of binding to two antigenic sites.

Other suitable methods known in the art for producing bifunctionalantibodies may also be used, which include those disclosed in Holligeret al., Proc. Nat'l Acad. Sci. USA, 90:6444-6448 (1993); de Kruif etal., J. Biol. Chem., 271:7630-7634 (1996); Coloma and Morrison, Nat.Biotechnol., 15:159-163 (1997); Muller et al., FEBS Lett., 422:259-264(1998); and Muller et al., FEBS Lett., 432:45-49 (1998), all of whichare incorporated herein by reference.

4. Methods of Detecting Protein Complexes

Another aspect of the present invention relates to methods for detectingthe protein complexes of the present invention, particularly fordetermining the concentration of a specific protein complex in a patientsample.

In one embodiment, the concentration of a protein complex having VAP-Aand one or more proteins selected from the group consisting of PPP1R3and GTR3 is determined in cells, tissue, or an organ of a patient. Forexample, the protein complex can be isolated or purified from a patientsample obtained from cells, tissue, or an organ of the patient and theamount thereof is determined. As described above, the protein complexcan be prepared from cells, tissue or organ samples bycoimmunoprecipitation using an antibody immunoreactive with aninteracting protein member, a bifunctional antibody that isimmunoreactive with two or more interacting protein members of theprotein complex, or preferably an antibody selectively immunoreactivewith the protein complex. When bifunctional antibodies or antibodiesimmunoreactive with only free interacting protein members are used,individual interacting protein members not complexed with other proteinsmay also be isolated along with the protein complex containing suchindividual proteins. However, they can be readily separated from theprotein complex using methods known in the art, e.g., size-basedseparation methods such as gel filtration, or by subtracting the proteincomplex from the mixture using an antibody specific against anotherindividual interacting protein member. Additionally, proteins in asample can be separated in a gel such as polyacrylamide gel andsubsequently immunoblotted using an antibody immunoreactive with theprotein complex.

Alternatively, the concentration of the protein complex can bedetermined in a sample without separation, isolation or purification.For this purpose, it is preferred that an antibody selectivelyimmunoreactive with the specific protein complex is used in animmunoassay. For example, immunocytochemical methods can be used. Otherwell known antibody-based techniques can also be used including, e.g.,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assays (IRMA), fluorescent immunoassays, protein Aimmunoassays, and immunoenzymatic assays (IEMA). See e.g., U.S. Pat.Nos. 4,376,110 and 4,486,530, both of which are incorporated herein byreference.

In addition, since a specific protein complex is formed from itsinteracting protein members, if one of the interacting protein membersis at a relatively low concentration in a patient, it may be reasonablyexpected that the concentration of the protein complex in the patientmay also be low. Therefore, the concentration of an individualinteracting protein member of a specific protein complex can bedetermined in a patient sample which can then be used as a reasonablyaccurate indicator of the concentration of the protein complex in thesample. For this purpose, antibodies against an individual interactingprotein member of a specific complex can be used in any one of themethods described above. In a preferred embodiment, the concentration ofeach of the interacting protein members of a protein complex isdetermined in a patient sample and the relative concentration of theprotein complex is then deduced.

In addition, the relative protein complex concentration in a patient canalso be determined by determining the concentration of the mRNA encodingan interacting protein member of the protein complex. Preferably, eachinteracting protein member's mRNA concentration in a patient sample isdetermined. For this purpose, methods for determining mRNA concentrationgenerally known in the art may all be used. Examples of such methodsinclude, e.g., Northern blot assay, dot blot assay, PCR assay(preferably quantitative PCR assay), in situ hybridization assay, andthe like.

As discussed above, the interactions between VAP-A and the proteinsPPP1R3 and GTR3 suggest that these proteins and/or the protein complexesformed by such proteins may be involved in common biological processesand disease pathways. In addition, the interactions between VAP-A andPPP1R3 and GTR3 under physiological conditions may lead to the formationof protein complexes in vivo that contain VAP-A and one or more of theVAP-A-interacting proteins. The protein complexes are expected tomediate the functions and biological activities of VAP-A and PPP1R3 andGTR3. For example, VAP-A and the VAP-A-interacting proteins may beinvolved in docking and fusion of membrane vesicles to target organellemembranes and cellular uptake of glucose and associated with diseasesand disorders such as diabetes, obesity, ischemia, and insulinresistance. Thus, aberrations in the concentration and/or activity ofthe protein complexes and/or the proteins such as VAP-A and theVAP-A-interacting proteins may result in diseases or disorders such asdiabetes, obesity, ischemia, and insulin resistance. Thus, theaberration in the protein complexes or the individual proteins and thedegree of the aberration may be indicators for the diseases ordisorders. These aberrations may be used as parameters for classifyingand/or staging one of the above-described diseases. In addition, theymay also be indicators for patients' response to a drug therapy.

Association between a physiological state (e.g., physiological disorder,predisposition to the disorder, a disease state, response to a drugtherapy, or other physiological phenomena or phenotypes) and a specificaberration in a protein complex of the present invention or anindividual interacting member thereof can be readily determined bycomparative analysis of the protein complex and/or the interactingmembers thereof in a normal population and an abnormal or affectedpopulation. Thus, for example, one can study the concentration,localization and distribution of a particular protein complex, mutationsin the interacting protein members of the protein complex, and/or thebinding affinity between the interacting protein members in both anormal population and a population affected with a particularphysiological disorder described above. The study results can becompared and analyzed by statistical means. Any detected statisticallysignificant difference in the two populations would indicate anassociation. For example, if the concentration of the protein complex isstatistically significantly higher in the affected population than inthe normal population, then it can be reasonably concluded that higherconcentration of the protein complex is associated with thephysiological disorder.

Thus, once an association is established between a particular type ofaberration in a particular protein complex of the present invention orin an interacting protein member thereof and a physiological disorder ordisease or predisposition to the physiological disorder or disease, thenthe particular physiological disorder or disease or predisposition tothe physiological disorder or disease can be diagnosed or detected bydetermining whether a patient has the particular aberration.

Accordingly, the present invention also provides a method for diagnosingin a patient a disease or physiological disorder, or a predisposition tothe disease or disorder, such as diabetes, obesity, ischemia, andinsulin resistance by determining whether there is any aberration in thepatient with respect to a protein complex having a first protein whichis VAP-A interacting with a second protein selected from the groupconsisting of PPP1R3 and GTR3. The same protein complex is analyzed in anormal individual and is compared with the results obtained in thepatient. In this manner, any protein complex aberration in the patientcan be detected. As used herein, the term “aberration” when used in thecontext of protein complexes of the present invention means anyalterations of a protein complex including increased or decreasedconcentration of the protein complex in a particular cell or tissue ororgan or the total body, altered localization of the protein complex incellular compartments or in locations of a tissue or organ, changes inbinding affinity of an interacting protein member of the proteincomplex, mutations in an interacting protein member or the gene encodingthe protein, and the like. As will be apparent to a skilled artisan, theterm “aberration” is used in a relative sense. That is, an aberration isrelative to a normal condition.

As used herein, the term “diagnosis” means detecting a disease ordisorder or determining the stage or degree of a disease or disorder.The term “diagnosis” also encompasses detecting a predisposition to adisease or disorder, determining the therapeutic effect of a drugtherapy, or predicting the pattern of response to a drug therapy orxenobiotics. The diagnosis methods of the present invention may be usedindependently, or in combination with other diagnosing and/or stagingmethods known in the medical art for a particular disease or disorder.

Thus, in one embodiment, the method of diagnosis is conducted bydetecting, in a patient, the concentrations of one or more proteincomplexes of the present invention using any one of the methodsdescribed above, and determining whether the patient has an aberrantconcentration of the protein complexes.

The diagnosis may also be based on the determination of theconcentrations of one or more interacting protein members (at theprotein, cDNA or mRNA level) of a protein complex of the presentinvention. An aberrant concentration of an interacting protein membermay indicate a physiological disorder or a predisposition to aphysiological disorder.

In another embodiment, the method of diagnosis comprises determining, ina patient, the cellular localization, or tissue or organ distribution ofa protein complex of the present invention and determining whether thepatient has an aberrant localization or distribution of the proteincomplex. For example, immunocytochemical or immunohistochemical assayscan be performed on a cell, tissue or organ sample from a patient usingan antibody selectively immunoreactive with a protein complex of thepresent invention. Antibodies immunoreactive with both an individualinteracting protein member and a protein complex containing the proteinmember may also be used, in which case it is preferred that antibodiesimmunoreactive with other interacting protein members are also used inthe assay. In addition, nucleic acid probes may also be used in in situhybridization assays to detect the localization or distribution of themRNAs encoding the interacting protein members of a protein complex.Preferably, the mRNA encoding each interacting protein member of aprotein complex is detected concurrently.

In yet another embodiment, the method of diagnosis of the presentinvention comprises detecting any mutations in one or more interactingprotein members of a protein complex of the present invention. Inparticular, it is desirable to determine whether the interacting proteinmembers have any mutations that will lead to, or are associated with,changes in the functional activity of the proteins or changes in theirbinding affinity to other interacting protein members in forming aprotein complex of the present invention. Examples of such mutationsinclude but are not limited to, e.g., deletions, insertions andrearrangements in the genes encoding the protein members, and nucleotideor amino acid substitutions and the like. In a preferred embodiment, thedomains of the interacting protein members that are responsible for theprotein-protein interactions, and lead to protein complex formation, arescreened to detect any mutations therein. For example, genomic DNA orcDNA encoding an interacting protein member can be prepared from apatient sample, and sequenced. The thus obtained sequence may becompared with known wild-type sequences to identify any mutations.Alternatively, an interacting protein member may be purified from apatient sample and analyzed by protein sequencing or mass spectrometryto detect any amino acid sequence changes. Any methods known in the artfor detecting mutations may be used, as will be apparent to skilledartisans apprised of the present disclosure.

In another embodiment, the method of diagnosis includes determining thebinding constant of the interacting protein members of one or moreprotein complexes. For example, the interacting protein members can beobtained from a patient by direct purification or by recombinantexpression from genomic DNAs or cDNAs prepared from a patient sampleencoding the interacting protein members. Binding constants representthe strength of the protein-protein interaction between the interactingprotein members in a protein complex. Thus, by measuring bindingconstant, subtle aberration in binding affinity may be detected.

A number of methods known in the art for estimating and determiningbinding constants in protein-protein interactions are reviewed inPhizicky and Fields, et al., Microbiol. Rev., 59:94-123 (1995), which isincorporated herein by reference. For example, protein affinitychromatography may be used. First, columns are prepared with differentconcentrations of an interacting protein member which is covalentlybound to the columns. Then a preparation of an interacting proteinpartner is run through the column and washed with buffer. Theinteracting protein partner bound to the interacting protein memberlinked to the column is then eluted. Binding constant is then estimatedbased on the concentrations of the bound protein and the eluted protein.Alternatively, the method of sedimentation through gradients monitorsthe rate of sedimentation of a mixture of proteins through gradients ofglycerol or sucrose. At concentrations above the binding constant,proteins can sediment as a protein complex. Thus, binding constant canbe calculated based on the concentrations. Other suitable methods knownin the art for estimating binding constant include but are not limitedto gel filtration column such as nonequilibrium “small-zone” gelfiltration columns (See e.g., Gill et al., J. Mol. Biol., 220:307-324(1991)), the Hummel-Dreyer method of equilibrium gel filtration (Seee.g., Hummel and Dreyer, Biochim. Biophys. Acta, 63:530-532 (1962)) andlarge-zone equilibrium gel filtration (See e.g., Gilbert and Kellett, J.Biol. Chem., 246:6079-6086 (1971)), sedimentation equilibrium (See e.g.,Rivas and Minton, Trends Biochem., 18:284-287 (1993)), fluorescencemethods such as fluorescence spectrum (See e.g., Otto-Bruc et al,Biochemistry, 32:8632-8645 (1993)) and fluorescence polarization oranisotropy with tagged molecules (See e.g., Weiel and Hershey,Biochemistry, 20:5859-5865 (1981)), solution equilibrium measured withimmobilized binding protein (See e.g., Nelson and Long, Biochemistry,30:2384-2390 (1991)), and surface plasmon resonance (See e.g., Panayotouet al., Mol. Cell. Biol., 13:3567-3576 (1993)).

In another embodiment, the diagnosis method of the present inventioncomprises detecting protein-protein interactions in functional assaysystems such as the yeast two-hybrid system. Accordingly, to determinethe protein-protein interaction between two interacting protein membersthat normally form a protein complex in normal individuals, cDNAsencoding the interacting protein members can be isolated from a patientto be diagnosed. The thus cloned cDNAs or fragments thereof can besubcloned into vectors for use in yeast two-hybrid system. Preferably areverse yeast two-hybrid system is used such that failure of interactionbetween the proteins may be positively detected. The use of yeasttwo-hybrid system or other systems for detecting protein-proteininteractions is known in the art and is described below in Section5.3.1.

A kit may be used for conducting the diagnosis methods of the presentinvention. Typically, the kit should contain, in a carrier orcompartmentalized container, reagents useful in any of theabove-described embodiments of the diagnosis method. The carrier can bea container or support, in the form of, e.g., bag, box, tube, rack, andis optionally compartmentalized. The carrier may define an enclosedconfinement for safety purposes during shipment and storage. In oneembodiment, the kit includes an antibody selectively immunoreactive witha protein complex of the present invention. In addition, antibodiesagainst individual interacting protein members of the protein complexesmay also be included. The antibodies may be labeled with a detectablemarker such as radioactive isotopes, or enzymatic or fluorescencemarkers. Alternatively secondary antibodies such as labeled anti-IgG andthe like may be included for detection purposes. Optionally, the kit caninclude one or more of the protein complexes of the present inventionprepared or purified from a normal individual or an individual afflictedwith a physiological disorder associated with an aberration in theprotein complexes or an interacting protein member thereof. In addition,the kit may further include one or more of the interacting proteinmembers of the protein complexes of the present invention prepared orpurified from a normal individual or an individual afflicted with aphysiological disorder associated with an aberration in the proteincomplexes or an interacting protein member thereof. Suitableoligonucleotide primers useful in the amplification of the genes orcDNAs for the interacting protein members may also be provided in thekit. In particular, in a preferred embodiment, the kit includes a firstoligonucleotide selectively hybridizable to the mRNA or cDNA encodingVAP-A and a second oligonucleotide selectively hybridizable to the mRNAor cDNA encoding a protein selected from the group consisting of PPP1R3and GTR3. Additional oligonucleotides hybridizing to a region of thegene encoding VAP-A and/or a region of the gene(s) encoding one or moreVAP-A-interacting proteins, as identified in the present invention, mayalso be included. Such oligonucleotides may be used as PCR primers for,e.g., quantitative PCR amplification of mRNAs encoding VAP-A and aninteracting partner thereof, or as hybridizing probes for detecting themRNAs. The oligonucleotides may have a length of from about 8nucleotides to about 100 nucleotides, preferably from about 12 to about50 nucleotides, and more preferably from about 15 to about 30nucleotides. In addition, the kit may also contain oligonucleotides thatcan be used as hybridization probes for detecting the cDNAs or mRNAsencoding the interacting protein members. Preferably, instructions forusing the kit or reagents contained therein are also included in thekit.

5. Use of Protein Complexes or Interacting Protein Members thereof inScreening Assays for Modulators

The protein complexes of the present invention and VAP-A andVAP-A-interacting proteins such as PPP1R3 and GTR3 can also be used inscreening assays to identify modulators of the protein complexes, VAP-A,and/or the VAP-A-interacting proteins. In addition, homologues,derivatives and fragments of VAP-A and homologues, derivatives andfragments of the VAP-A-interacting proteins may also be used in suchscreening assays. As used herein, the term “modulator” encompasses anycompounds that can cause any forms of alteration of the biologicalactivities or functions of the proteins or protein complexes, including,e.g., enhancing or reducing their biological activities, increasing ordecreasing their stability, altering their affinity or specificity tocertain other biological molecules, etc. In addition, the term“modulator” as used herein also includes any compounds that simply bindVAP-A, VAP-A-interacting proteins, and/or the proteins complexes of thepresent invention. For example, a modulator can be an “interactionantagonist” capable of interfering with or disrupting or dissociatingprotein-protein interaction between VAP-A or a homologue, fragment orderivative thereof and one or more proteins selected from the groupconsisting of PPP1R3 and GTR3 or a homologue, fragment or derivativethereof. A modulator can also be an “interaction agonist” that initiatesor strengthens the interaction between the protein members of a proteincomplex of the present invention, or homologues, fragments orderivatives thereof.

Accordingly, the present invention provides screening methods forselecting modulators of VAP-A, or a mutant form thereof, aVAP-A-interacting protein selected from the group consisting of PPP1R3and GTR3, or a mutant form thereof, or a protein complex formed betweenVAP-A, or a mutant form thereof, and one or more of theVAP-A-interacting proteins, or a mutant forms thereof. Screening methodsare also provided for selecting modulators of VAP-A homologues,derivatives or fragments, or homologues, derivatives or fragments of aVAP-A-interacting protein, or a protein complex formed between a VAP-Ahomologue, derivative or fragment and a homologue or derivative orfragment of a VAP-A-interacting protein, or proteins.

The selected compounds can be tested for their ability to modulate(interfere with or strengthen) the interaction between the interactingpartners within the protein complexes of the present invention. Inaddition, the compounds can also be further tested for their ability tomodulate (inhibit or enhance) cellular functions such as docking andfusion of membrane vesicles to target organelle membranes and cellularuptake of glucose in cells as well as their effectiveness in treatingdiseases such as diabetes, obesity, ischemia, and insulin resistance.

The modulators selected in accordance with the screening methods of thepresent invention can be effective in modulating the functions oractivities of VAP-A, a VAP-A-interacting protein, or the proteincomplexes of the present invention. For example, compounds capable ofbinding to the protein complexes may be capable of modulating thefunctions of the protein complexes. Additionally, compounds thatinterfere with, weaken, dissociate or disrupt, or alternatively,initiate, facilitate or stabilize the protein-protein interactionbetween the interacting protein members of the protein complexes canalso be effective in modulating the functions or activities of theprotein complexes. Thus, the compounds identified in the screeningmethods of the present invention can be made into therapeutically orprophylactically effective drugs for preventing or amelioratingdiseases, disorders or symptoms caused by or associated with aberrationsin the protein complexes or VAP-A or the VAP-A-interacting proteins ofthe present invention. Alternatively, they may be used as leads to aidthe design and identification of therapeutically or prophylacticallyeffective compounds for diseases, disorders or symptoms caused by orassociated with aberrations in the protein complexes or VAP-A or theVAP-A-interacting proteins of the present invention. The proteincomplexes and/or interacting protein members thereof in accordance withthe present invention can be used in any of a variety of drug screeningtechniques. Drug screening can be performed as described herein or usingwell-known techniques, such as those described in U.S. Pat. Nos.5,800,998 and 5,891,628, both of which are incorporated herein byreference.

5.1. Test Compounds

Any test compounds may be screened in the screening assays of thepresent invention to select modulators of VAP-A, a VAP-A-containingprotein complex and/or a VAP-A-interacting protein of the presentinvention. By the term “selecting” or “select” compounds it is intendedto encompass both (a) choosing compounds from a group previously unknownto be modulators of VAP-A, a VAP-A-containing protein complex and/or aVAP-A-interacting protein of the present invention, and (b) testingcompounds that are known to be capable of binding, or modulating thefunctions and activities of, VAP-A, a VAP-A-containing protein complexand/or a VAP-A-interacting protein of the present invention. Both typesof compounds are generally referred to herein as “test compounds.” Thetest compounds may include, by way of example, proteins (e.g.,antibodies, small peptides, artificial or natural proteins), nucleicacids, and derivatives, mimetics and analogs thereof, and small organicmolecules having a molecular weight of no greater than 10,000 daltons,more preferably less than 5,000 daltons. Preferably, the test compoundsare provided in library formats known in the art, e.g., in chemicallysynthesized libraries, recombinantly expressed libraries (e.g., phagedisplay libraries), and in vitro translation-based libraries (e.g.,ribosome display libraries).

For example, the screening assays of the present invention can be usedin the antibody production processes described in Section 3 to selectantibodies with desirable specificities. Various forms antibodies orderivatives thereof may be screened, including but not limited to,polyclonal antibodies, monoclonal antibodies, bifunctional antibodies,chimeric antibodies, single chain antibodies, antibody fragments such asFv fragments, single-chain Fv fragments (scFv), Fab′ fragments, andF(ab′)₂ fragments, and various modified forms of antibodies such ascatalytic antibodies, and antibodies conjugated to toxins or drugs, andthe like. The antibodies can be of any types such as IgG, IgE, IgA, orIgM. Humanized antibodies are particularly preferred. Preferably, thevarious antibodies and antibody fragments may be provided in librariesto allow large-scale high throughput screening. For example, expressionlibraries expressing antibodies or antibody fragments may be constructedby a method disclosed, e.g., in Huse et al., Science, 246:1275-1281(1989), which is incorporated herein by reference. Single-chain Fv(scfv) antibodies are of particular interest in diagnostic andtherapeutic applications. Methods for providing antibody libraries arealso provided in U.S. Pat. Nos. 6,096,551; 5,844,093; 5,837,460;5,789,208; and 5,667,988, all of which are incorporated herein byreference.

Peptidic test compounds may be peptides having L-amino acids and/orD-amino acids, phosphopeptides, and other types of peptides. Thescreened peptides can be of any size, but preferably have less thanabout 50 amino acids. Smaller peptides are easier to deliver into apatient's body. Various forms of modified peptides may also be screened.Like antibodies, peptides can also be provided in, e.g., combinatoriallibraries. See generally, Gallop et al., J. Med. Chem., 37:1233-1251(1994). Methods for making random peptide libraries are disclosed in,e.g., Devlin et al., Science, 249:404-406 (1990). Other suitable methodsfor constructing peptide libraries and screening peptides therefrom aredisclosed in, e.g., Scott and Smith, Science, 249:386-390 (1990); Moranet al., J. Am. Chem. Soc., 117:10787-10788 (1995) (a library ofelectronically tagged synthetic peptides); Stachelhaus et al., Science,269:69-72 (1995); U.S. Pat. Nos. 6,156,511; 6,107,059; 6,015,561;5,750,344; 5,834,318; 5,750,344, all of which are incorporated herein byreference. For example, random-sequence peptide phage display librariesmay be generated by cloning synthetic oligonucleotides into the gene IIIor gene VIII of an E. coli. filamentous phage. The thus generated phagecan propagate in E.'coli. and express peptides encoded by theoligonucleotides as fusion proteins on the surface of the phage. Scottand Smith, Science, 249:368-390 (1990). Alternatively, the “peptides onplasmids” method may also be used to form peptide libraries. In thismethod, random peptides may be fused to the C-terminus of the E. coli.Lac repressor by recombinant technologies and expressed from a plasmidthat also contains Lac repressor-binding sites. As a result, the peptidefusions bind to the same plasmid that encodes them.

Small organic or inorganic non-peptide non-nucleotide compounds arepreferred test compounds for the screening assays of the presentinvention. They too can be provided in a library format. See generally,Gordan et al. J. Med. Chem., 37:1385-1401 (1994). For example,benzodiazepine libraries are provided in Bunin and Ellman, J. Am. Chem.Soc., 114:10997-10998 (1992), which is incorporated herein by reference.A method for constructing and screening peptoid libraries are disclosedin Simon et al., Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992).Methods for the biosynthesis of novel polyketides in a library formatare described in McDaniel et al, Science, 262:1546-1550 (1993) and Kaoet al., Science, 265:509-512 (1994). Various libraries of small organicmolecules and methods of construction thereof are disclosed in U.S. Pat.No. 6,162,926 (multiply-substituted fullerene derivatives); U.S. Pat.No. 6,093,798 (hydroxamic acid derivatives); U.S. Pat. No. 5,962,337(combinatorial 1,4-benzodiazepin-2, 5-dione library); U.S. Pat. No.5,877,278 (Synthesis of N-substituted oligomers); U.S. Pat. No.5,866,341 (compositions and methods for screening drug libraries); U.S.Pat. No. 5,792,821 (polymerizable cyclodextrin derivatives); U.S. Pat.No. 5,766,963 (hydroxypropylamine library); and U.S. Pat. No. 5,698,685(morpholino-subunit combinatorial library), all of which areincorporated herein by reference.

Other compounds such as oligonucleotides and peptide nucleic acids(PNA), and analogs and derivatives thereof may also be screened toidentify clinically useful compounds. Combinatorial libraries ofoligonucleotides are also known in the art. See Gold et al., J. Biol.Chem., 270:13581-13584 (1995).

5.2. In vitro Screening Assays

The test compounds may be screened in an in vitro assay to identifycompounds capable of binding the protein complexes or interactingprotein members thereof in accordance with the present invention. Forthis purpose, a test compound is contacted with a protein complex or aninteracting protein member thereof under conditions and for a timesufficient to allow specific interaction between the test compound andthe target components to occur and thus binding of the compound to thetarget forming a complex. Subsequently, the binding event is detected.

Various screening techniques known in the art may be used in the presentinvention. The protein complexes and the interacting protein membersthereof may be prepared by any suitable methods, e.g., by recombinantexpression and purification. The protein complexes and/or interactingprotein members thereof (both are referred to as “target” hereinafter inthis section) may be free in solution. A test compound may be mixed witha target forming a liquid mixture. The compound may be labeled with adetectable marker. Upon mixing under suitable conditions, the bindingcomplex having the compound and the target may be co-immunoprecipitatedand washed. The compound in the precipitated complex may be detectedbased on the marker on the compound.

In a preferred embodiment, the target is immobilized on a solid supportor on a cell surface. Preferably, the target can be arrayed into aprotein microchip in a method described in Section 2.3. For example, atarget may be immobilized directly onto a microchip substrate such asglass slides or onto a multi-well plates using non-neutralizingantibodies, i.e., antibodies capable of binding to the target but do notsubstantially affect its biological activities. To affect the screening,test compounds can be contacted with the immobilized target to allowbinding to occur to form complexes under standard binding assayconditions. Either the targets or test compounds are labeled with adetectable marker using well-known labeling techniques. For example,U.S. Pat. No. 5,741,713 discloses combinatorial libraries of biochemicalcompounds labeled with NMR active isotopes. To identify bindingcompounds, one may measure the formation of the target-test compoundcomplexes or kinetics for the formation thereof. When combinatoriallibraries of organic non-peptide non-nucleic acid compound are screened,it is preferred that labeled or encoded (or “tagged”) combinatoriallibraries are used to allow rapid decoding of lead structures. This isespecially important because, unlike biological libraries, individualcompounds found in chemical libraries cannot be amplified byself-amplification. Tagged combinatorial libraries are provided in,e.g., Borchardt and Still, J. Am. Chem. Soc., 116:373-374 (1994) andMoran et al., J. Am. Chem. Soc., 117:10787-10788 (1995), both of whichare incorporated herein by reference.

Alternatively, the test compounds can be immobilized on a solid support,e.g., forming a microarray of test compounds. The target protein orprotein complex is then contacted with the test compounds. The targetmay be labeled with any suitable detection marker. For example, thetarget may be labeled with radioactive isotopes or fluorescence markerbefore binding reaction occurs. Alternatively, after the bindingreactions, antibodies that are immunoreactive with the target and arelabeled with radioactive materials, fluorescence markers, enzymes, orlabeled secondary anti-Ig antibodies may be used to detect any boundtarget thus identifying the binding compound. One example of thisembodiment is the protein probing method. That is, the target providedin accordance with the present invention is used as a probe to screenexpression libraries of proteins or random peptides. The expressionlibraries can be phage display libraries, in vitro translation-basedlibraries, or ordinary expression cDNA libraries. The libraries may beimmobilized on a solid support such as nitrocellulose filters. See e.g.,Sikela and Hahn, Proc. Natl. Acad. Sci. USA, 84:3038-3042 (1987). Theprobe may be labeled with a radioactive isotope or a fluorescencemarker. Alternatively, the probe can be biotinylated and detected with astreptavidin-alkaline phosphatase conjugate. More conveniently, thebound probe may be detected with an antibody.

In yet another embodiment, a known ligand capable of binding to thetarget can be used in competitive binding assays. Complexes between theknown ligand and the target can be formed and then contacted with testcompounds. The ability of a test compound to interfere with theinteraction between the target and the known ligand is measured. Oneexemplary ligand is an antibody capable of specifically binding thetarget. Particularly, such an antibody is especially useful foridentifying peptides that share one or more antigenic determinants ofthe target protein complex or interacting protein members thereof.

In a specific embodiment, a protein complex used in the screening assayincludes a hybrid protein as described in Section 2.1, which is formedby fusion of two interacting protein members or fragments or interactiondomains thereof. The hybrid protein may also be designed such that itcontains a detectable epitope tag fused thereto. Suitable examples ofsuch epitope tags include sequences derived from, e.g., influenza virushemagglutinin (HA), Simian Virus 5 (V5), polyhistidine (6xHis), c-myc,lacZ, GST, and the like.

Test compounds may be also screened in an in vitro assay to identifycompounds capable of dissociating the protein complexes identified inaccordance with the present invention. Thus, for example, aVAP-A-containing protein complex can be contacted with a test compoundand the protein complex can be detected. Conversely, test compounds mayalso be screened to identify compounds capable of enhancing theinteraction between VAP-A and a VAP-A-interacting protein or stabilizingthe protein complex formed by the two or more proteins.

The assay can be conducted in similar manners as the binding assaysdescribed above. For example, the presence or absence of a particularprotein complex can be detected by an antibody selectivelyimmunoreactive with the protein complex. Thus, after incubation of theprotein complex with a test compound, an immunoprecipitation assay canbe conducted with the antibody. If the test compound disrupts theprotein complex, then the amount of immunoprecipitated protein complexin this assay will be significantly less than that in a control assay inwhich the same protein complex is not contacted with the test compound.Similarly, two proteins the interaction between which is to be enhancedmay be incubated together with a test compound. Thereafter, a proteincomplex may be detected by the selectively immunoreactive antibody. Theamount of protein complex may be compared to that formed in the absenceof the test compound. Various other detection methods may be suitable inthe dissociation assay, as will be apparent to skilled artisan apprisedof the present disclosure.

5.3. In vivo Screening Assays

Test compounds can also be screened in any in vivo assays to selectmodulators of the protein complexes or interacting protein membersthereof in accordance with the present invention. For example, any invivo assays known in the art to be useful in identifying compoundscapable of strengthening or interfering with the stability of theprotein complexes of the present invention may be used.

5.3.1. Two-Hybrid Assays

In a preferred embodiment, one of the yeast two-hybrid systems or theiranalogous or derivative forms is used. Examples of suitable two-hybridsystems known in the art include, but are not limited to, thosedisclosed in U.S. Pat. Nos. 5,283,173; 5,525,490; 5,585,245; 5,637,463;5,695,941; 5,733,726; 5,776,689; 5,885,779; 5,905,025; 6,037,136;6,057,101; 6,114,111; and Bartel and Fields, eds., The Yeast Two-HybridSystem, Oxford University Press, New York, N.Y., 1997, all of which areincorporated herein by reference.

Typically, in a classic transcription-based two-hybrid assay, twochimeric genes are prepared encoding two fusion proteins: one contains atranscription activation domain fused to an interacting protein memberof a protein complex of the present invention or an interaction domainor fragment of the interacting protein member, while the other fusionprotein includes a DNA binding domain fused to another interactingprotein member of the protein complex or a fragment or interactiondomain thereof. For the purpose of convenience, the two interactingprotein members, fragments or interaction domains thereof are referredto as “bait fusion protein” and “prey fusion protein,” respectively. Thechimeric genes encoding the fusion proteins are termed “bait chimericgene” and “prey chimeric gene,” respectively. Typically, a “bait vector”and a “prey vector” are provided for the expression of a bait chimericgene and a prey chimeric gene, respectively.

5.3.1.1. Vectors

Many types of vectors can be used in a transcription-based two-hybridassay. Methods for the construction of bait vectors and prey vectorsshould be apparent to skilled artisans in the art apprised of thepresent disclosure. See generally, Current Protocols in MolecularBiology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & WileyInterscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRL Press,Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods in Enzymology153:516-544 (1987); The Molecular Biology of the Yeast Saccharomyces,Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982;and Rothstein in DNA Cloning: A Practical Approach, Vol. 11, Ed. DMGlover, IRL Press, Wash., D.C., 1986.

Generally, the bait and prey vectors include an expression cassettehaving a promoter operably linked to a chimeric gene for thetranscription of the chimeric gene. The vectors may also include anorigin of DNA replication for the replication of the vectors in hostcells and a replication origin for the amplification of the vectors in,e.g., E. coli, and selection marker(s) for selecting and maintainingonly those host cells harboring the vectors. Additionally, theexpression cassette preferably also contains inducible elements, whichfunction to control the expression of a chimeric gene. Making theexpression of the chimeric genes inducible and controllable isespecially important in the event that the fusion proteins or componentsthereof are toxic to the host cells. Other regulatory sequences such astranscriptional enhancer sequences and translation regulation sequences(e.g., Shine-Dalgarno sequence) can also be included in the expressioncassette. Termination sequences such as the bovine growth hormone, SV40,lacZ and AcMNPV polyhedral polyadenylation signals may also be operablylinked to a chimeric gene in the expression cassette. An epitope tagcoding sequence for detection and/or purification of the fusion proteinscan also be operably linked to the chimeric gene in the expressioncassette. Examples of useful epitope tags include, but are not limitedto, influenza virus hemagglutinin (HA), Simian Virus 5 (V5),polyhistidine (6xHis), c-myc, lacZ, GST, and the like. Proteins withpolyhistidine tags can be easily detected and/or purified with Niaffinity columns, while specific antibodies to many epitope tags aregenerally commercially available. The vectors can be introduced into thehost cells by any techniques known in the art, e.g., by direct DNAtransformation, microinjection, electroporation, viral infection,lipofection, gene gun, and the like. The bait and prey vectors can bemaintained in host cells in an extrachromosomal state, i.e., asself-replicating plasmids or viruses. Alternatively, one or both vectorscan be integrated into chromosomes of the host cells by conventionaltechniques such as selection of stable cell lines or site-specificrecombination.

The in vivo assays of the present invention can be conducted in manydifferent host cells, including but not limited to bacteria, yeastcells, plant cells, insect cells, and mammalian cells. A skilled artisanwill recognize that the designs of the vectors can vary with the hostcells used. In one embodiment, the assay is conducted in prokaryoticcells such as Escherichia coli, Salmonella, Klebsiella, Pseudomonas,Caulobacter, and Rhizobium. Suitable origins of replication for theexpression vectors useful in this embodiment of the present inventioninclude, e.g., the ColE1, pSC101, and M13 origins of replication.Examples of suitable promoters include, for example, the T7 promoter,the lacZ promoter, and the like. In addition, inducible promoters arealso useful in modulating the expression of the chimeric genes. Forexample, the lac operon from bacteriophage lambda plac5 is well known inthe art and is inducible by the addition of IPTG to the growth medium.Other known inducible promoters useful in a bacteria expression systeminclude pL of bacteriophage λ, the trp promoter, and hybrid promoterssuch as the tac promoter, and the like.

In addition, selection marker sequences for selecting and maintainingonly those prokaryotic cells expressing the desirable fusion proteinsshould also be incorporated into the expression vectors. Numerousselection markers including auxotrophic markets and antibioticresistance markers are known in the art and can all be useful forpurposes of this invention. For example, the bla gene, which confersampicillin resistance, is the most commonly used selection marker inprokaryotic expression vectors. Other suitable markers include genesthat confer neomycin, kanamycin, or hygromycin resistance to the hostcells. In fact, many vectors are commercially available from vendorssuch as Invitrogen Corp. of Carlsbad, Calif., Clontech Corp. of PaloAlto, Calif., and Stratagene Corp. of La Jolla, Calif., and PromegaCorp. of Madison, Wis. These commercially available vectors, e.g.,pBR322, pSPORT, pBluescriptIISK, pcDNAI, and pcDNAII all have a multiplecloning site into which the chimeric genes of the present invention canbe conveniently inserted using conventional recombinant techniques. Theconstructed expression vectors can be introduced into host cells byvarious transformation or transfection techniques generally known in theart.

In another embodiment, mammalian cells are used as host cells for theexpression of the fusion proteins and detection of protein-proteininteractions. For this purpose, virtually any mammalian cells can beused including normal tissue cells, stable cell lines, and transformedtumor cells. Conveniently, mammalian cell lines such as CHO cells,Jurkat T cells, NIH 3T3 cells, HEK-293 cells, CV-1 cells, COS-1 cells,HeLa cells, VERO cells, MDCK cells, WI38 cells, and the like are used.Mammalian expression vectors are well known in the art and many arecommercially available. Examples of suitable promoters for thetranscription of the chimeric genes in mammalian cells include viraltranscription promoters derived from adenovirus, simian virus 40 (SV40)(e.g., the early and late promoters of SV40), Rous sarcoma virus (RSV),and cytomegalovirus (CMV) (e.g., CMV immediate-early promoter), humanimmunodeficiency virus (HIV) (e.g., long terminal repeat (LTR)),vaccinia virus (e.g., 7.5K promoter), and herpes simplex virus (HSV)(e.g., thymidine kinase promoter). Inducible promoters can also be used.Suitable inducible promoters include, for example, the tetracyclineresponsive element (TRE) (See Gossen et al., Proc. Natl. Acad. Sci. USA,89:5547-5551 (1992)), metallothionein IIA promoter, ecdysone-responsivepromoter, and heat shock promoters. Suitable origins of replication forthe replication and maintenance of the expression vectors in mammaliancells include, e.g., the Epstein Barr origin of replication in thepresence of the Epstein Barr nuclear antigen (see Sugden et al., Mole.Cell. Biol., 5:410-413 (1985)) and the SV40 origin of replication in thepresence of the SV40 T antigen (which is present in COS-1 and COS-7cells) (see Margolskee et al., Mole. Cell. Biol., 8:2837 (1988)).Suitable selection markers include, but are not limited to, genesconferring resistance to neomycin, hygromycin, zeocin, and the like.Many commercially available mammalian expression vectors may be usefulfor the present invention, including, e.g., pCEP4, pcDNAI, pIND,pSecTag2, pVAXI, pcDNA3.1, and pBI-EGFP, and pDisplay. The vectors canbe introduced into mammalian cells using any known techniques such ascalcium phosphate precipitation, lipofection, electroporation, and thelike. The bait vector and prey vector can be co-transformed into thesame cell or, alternatively, introduced into two different cells whichare subsequently fused together by cell fusion or other suitabletechniques.

Viral expression vectors, which permit introduction of recombinant genesinto cells by viral infection, can also be used for the expression ofthe fusion proteins. Viral expression vectors generally known in the artinclude viral vectors based on adenovirus, bovine papilloma virus,murine stem cell virus (MSCV), MFG virus, and retrovirus. See Sarver, etal., Mol. Cell. Biol., 1: 486 (1981); Logan & Shenk, Proc. Natl. Acad.Sci. USA, 81:3655-3659 (1984); Mackett, et al., Proc. Natl. Acad. Sci.USA, 79:7415-7419 (1982); Mackett, et al., J. Virol., 49:857-864 (1984);Panicali, et al., Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); Cone& Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353 (1984); Mann etal., Cell, 33:153-159 (1993); Pear et al., Proc. Natl. Acad. Sci. USA,90:8392-8396 (1993); Kitamura et al., Proc. Natl. Acad. Sci. USA,92:9146-9150 (1995); Kinsella et al., Human Gene Therapy, 7:1405-1413(1996); Hofmann et al., Proc. Natl. Acad. Sci. USA, 93:5185-5190 (1996);Choate et al., Human Gene Therapy, 7:2247 (1996); WO 94/19478; Hawley etal., Gene Therapy, 1:136 (1994) and Rivere et al., Genetics, 92:6733(1995), all of which are incorporated by reference.

Generally, to construct a viral vector, a chimeric gene according to thepresent invention can be operably linked to a suitable promoter. Thepromoter-chimeric gene construct is then inserted into a non-essentialregion of the viral vector, typically a modified viral genome. Thisresults in a viable recombinant virus capable of expressing the fusionprotein encoded by the chimeric gene in infected host cells. Once in thehost cell, the recombinant virus typically is integrated into the genomeof the host cell. However, recombinant bovine papilloma virusestypically replicate and remain as extrachromosomal elements.

In another embodiment, the detection assays of the present invention areconducted in plant cell systems. Methods for expressing exogenousproteins in plant cells are well known in the art. See generally,Weissbach & Weissbach, Methods for Plant Molecular Biology, AcademicPress, NY, 1988; Grierson & Corey, Plant Molecular Biology, 2d Ed.,Blackie, London, 1988. Recombinant virus expression vectors based on,e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic virus (TMV) canall be used. Alternatively, recombinant plasmid expression vectors suchas Ti plasmid vectors and Ri plasmid vectors are also useful. Thechimeric genes encoding the fusion proteins of the present invention canbe conveniently cloned into the expression vectors and placed undercontrol of a viral promoter such as the 35S RNA and 19S RNA promoters ofCaMV or the coat protein promoter of TMV, or of a plant promoter, e.g.,the promoter of the small subunit of RUBISCO and heat shock promoters(e.g., soybean hsp17.5-E or hsp17.3-B promoters).

In addition, the in vivo assay of the present invention can also beconducted in insect cells, e.g., Spodoptera frugiperda cells, using abaculovirus expression system. Expression vectors and host cells usefulin this system are well known in the art and are generally availablefrom various commercial vendors. For example, the chimeric genes of thepresent invention can be conveniently cloned into a non-essential region(e.g., the polyhedrin gene) of an Autographa californica nuclearpolyhedrosis virus (AcNPV) vector and placed under control of an AcNPVpromoter (e.g., the polyhedrin promoter). The non-occluded recombinantviruses thus generated can be used to infect host cells such asSpodoptera frugiperda cells in which the chimeric genes are expressed.See U.S. Pat. No. 4,215,051.

In a preferred embodiment of the present invention, the fusion proteinsare expressed in a yeast expression system using yeasts such asSaccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, andSchizosaccharomyces pombe as host cells. The expression of recombinantproteins in yeasts is a well-developed field, and the techniques usefulin this respect are disclosed in detail in The Molecular Biology of theYeast Saccharomyces, Eds. Strathern et al., Vols. I and II, Cold SpringHarbor Press, 1982; Ausubel et al., Current Protocols in MolecularBiology, New York, Wiley, 1994; and Guthrie and Fink, Guide to YeastGenetics and Molecular Biology, in Methods in Enzymology, Vol. 194,1991, all of which are incorporated herein by reference. Sudbery, Curr.Opin. Biotech., 7:517-524 (1996) reviews the success in the art inexpressing recombinant proteins in various yeast species; the entirecontent and references cited therein are incorporated herein byreference. In addition, Bartel and Fields, eds., The Yeast Two-HybridSystem, Oxford University Press, New York, N.Y., 1997 contains extensivediscussions of recombinant expression of fusion proteins in yeasts inthe context of various yeast two-hybrid systems, and cites numerousrelevant references. These and other methods known in the art can all beused for purposes of the present invention. The application of suchmethods to the present invention should be apparent to a skilled artisanapprised of the present disclosure.

Generally, each of the two chimeric genes is included in a separateexpression vector (bait vector and prey vector). Both vectors can beco-transformed into a single yeast host cell. As will be apparent to askilled artisan, it is also possible to express both chimeric genes froma single vector. In a preferred embodiment, the bait vector and preyvector are introduced into two haploid yeast cells of opposite matingtypes, e.g., a-type and α-type, respectively. The two haploid cells canbe mated at a desired time to form a diploid cell expressing bothchimeric genes.

Generally, the bait and prey vectors for recombinant expression in yeastinclude a yeast replication origin such as the 2μ origin or the ARSH4sequence for the replication and maintenance of the vectors in yeastcells. Preferably, the vectors also have a bacteria origin ofreplication (e.g., ColE1) and a bacteria selection marker (e.g., amp^(R)marker, i.e., bla gene). Optionally, the CEN6 centromeric sequence isincluded to control the replication of the vectors in yeast cells. Anyconstitutive or inducible promoters capable of driving genetranscription in yeast cells may be employed to control the expressionof the chimeric genes. Such promoters are operably linked to thechimeric genes. Examples of suitable constitutive promoters include butare not limited to the yeast ADH1, PGK1, TEF2, GPD1, HIS3, and CYC1promoters. Example of suitable inducible promoters include but are notlimited to the yeast GAL1 (inducible by galactose), CUP1 (inducible byCu⁺⁺), and FUS1 (inducible by pheromone) promoters; the AOX/MOX promoterfrom H. polymorpha and P. Pastoris (repressed by glucose or ethanol andinduced by methanol); chimeric promoters such as those that contain LexAoperators (inducible by LexA-containing transcription factors); and thelike. Inducible promoters are preferred when the fusion proteins encodedby the chimeric genes are toxic to the host cells. If it is desirable,certain transcription repressing sequences such as the upstreamrepressing sequence (URS) from SPO13 promoter can be operably linked tothe promoter sequence, e.g., to the 5′ end of the promoter region. Suchupstream repressing sequences function to fine-tune the expression levelof the chimeric genes.

Preferably, a transcriptional termination signal is operably linked tothe chimeric genes in the vectors. Generally, transcriptionaltermination signal sequences derived from, e.g., the CYC1 and ADH1 genescan be used.

Additionally, it is preferred that the bait vector and prey vectorcontain one or more selectable markers for the selection and maintenanceof only those yeast cells that harbor one or both chimeric genes. Anyselectable markers known in the art can be used for purposes of thisinvention so long as yeast cells expressing the chimeric gene(s) can bepositively identified or negatively selected. Examples of markers thatcan be positively identified are those based on color assays, includingthe lacZ gene (which encodes β-galactosidase), the firefly luciferasegene, secreted alkaline phosphatase, horseradish peroxidase, the bluefluorescent protein (BFP), and the green fluorescent protein (GFP) gene(see Cubitt et al., Trends Biochem. Sci., 20:448-455 (1995)). Othermarkers allowing detection by fluorescence, chemiluminescence, UVabsorption, infrared radiation, and the like can also be used. Among themarkers that can be selected are auxotrophic markers including, but notlimited to, URA3, HIS3, TRP1, LEU2, LYS2, ADE2, and the like. Typically,for purposes of auxotrophic selection, the yeast host cells transformedwith bait vector and/or prey vector are cultured in a medium lacking aparticular nutrient. Other selectable markers are not based onauxotrophies, but rather on resistance or sensitivity to an antibioticor other xenobiotic. Examples of such markers include but are notlimited to chloramphenicol acetyl transferase (CAT) gene, which confersresistance to chloramphenicol; CAN1 gene, which encodes an argininepermease and thereby renders cells sensitive to canavanine (see Sikorskiet al., Meth. Enzymol., 194:302-318 (1991)); the bacterial kanamycinresistance gene (kan^(R)), which renders eukaryotic cells resistant tothe aminoglycoside G418 (see Wach et al., Yeast, 10:1793-1808 (1994));and CYH2 gene, which confers sensitivity to cycloheximide (see Sikorskiet al., Meth. Enzymol., 194:302-318 (1991)). In addition, the CUP1 gene,which encodes metallothionein and thereby confers resistance to copper,is also a suitable selection marker. Each of the above selection markersmay be used alone or in combination. One or more selection markers canbe included in a particular bait or prey vector. The bait vector andprey vector may have the same or different selection markers. Inaddition, the selection pressure can be placed on the transformed hostcells either before or after mating the haploid yeast cells.

As will be apparent, the selection markers used should complement thehost strains in which the bait and/or prey vectors are expressed. Inother words, when a gene is used as a selection marker gene, a yeaststrain lacking the selection marker gene (or having mutation in thecorresponding gene) should be used as host cells. Numerous yeast strainsor derivative strains corresponding to various selection markers areknown in the art. Many of them have been developed specifically forcertain yeast two-hybrid systems. The application and optionalmodification of such strains with respect to the present inventionshould be apparent to a skilled artisan apprised of the presentdisclosure. Methods for genetically manipulating yeast strains usinggenetic crossing or recombinant mutagenesis are well known in the art.See e.g., Rothstein, Meth. Enzymol., 101:202-211 (1983). By way ofexample, the following yeast strains are well known in the art, and canbe used in the present invention upon necessary modifications andadjustment:

L40 strain which has the genotype MATa his3Δ200 trp1-901 leu2-3,112 ade2LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lacZ;

EGY48 strain which has the genotype MATα trp1 his3 ura3 6ops-LEU2; and

MaV103 strain which has the genotype MATα ura3-52 leu2-3,112 trp1-901his3Δ200 ade2-101 gal4Δ gal80Δ SPAL10::URA3 GAL1::HIS3::lys2 (see Kumaret al., J. Biol. Chem. 272:13548-13554 (1997); Vidal et al., Proc. Natl.Acad. Sci. USA, 93:10315-10320 (1996)). Such strains are generallyavailable in the research community, and can also be obtained by simpleyeast genetic manipulation. See, e.g., The Yeast Two-Hybrid System,Bartel and Fields, eds., pages 173-182, Oxford University Press, NewYork, N.Y., 1997.

In addition, the following yeast strains are commercially available:

Y190 strain which is available from Clontech, Palo Alto, Calif. and hasthe genotype MATa gal4 gal80 his3Δ200 trp1-901 ade2-101 ura3-52 leu2-3,112 URA3::GAL1-lacZ LYS2::GAL1-HIS3 cyh^(r); and

YRG-2 Strain which is available from Stratagene, La Jolla, Calif. andhas the genotype MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901leu2-3, 112 gal4-542 gal80-538 LYS2::GAL1-HIS3 URA3::GAL1/CYC1-lacZ.

In fact, different versions of vectors and host strains speciallydesigned for yeast two-hybrid system analysis are available in kits fromcommercial vendors such as Clontech, Palo Alto, Calif. and Stratagene,La Jolla, Calif., all of which can be modified for use in the presentinvention.

5.3.1.2. Reporters

Generally, in a transcription-based two-hybrid assay, the interactionbetween a bait fusion protein and a prey fusion protein brings theDNA-binding domain and the transcription-activation domain intoproximity forming a functional transcriptional factor that acts on aspecific promoter to drive the expression of a reporter protein. Thetranscription activation domain and the DNA-binding domain may beselected from various known transcriptional activators, e.g., GAL4,GCN4, ARD1, the human estrogen receptor, E. coli LexA protein, herpessimplex virus VP16 (Triezenberg et al., Genes Dev. 2:718-729 (1988)),the E. coli B42 protein (acid blob, see Gyuris et al., Cell, 75:791-803(1993)), NF-κB p65, and the like. The reporter gene and the promoterdriving its transcription typically are incorporated into a separatereporter vector. Alternatively, the host cells are engineered to containsuch a promoter-reporter gene sequence in their chromosomes. Thus, theinteraction or lack of interaction between two interacting proteinmembers of a protein complex can be determined by detecting or measuringchanges in the reporter in the assay system. Although the reporters andselection markers can be of similar types and used in a similar mannerin the present invention, the reporters and selection markers should becarefully selected in a particular detection assay such that they aredistinguishable from each other and do not interfere with each other'sfunction.

Many different types reporters are useful in the screening assays. Forexample, a reporter protein may be a fusion protein having an epitopetag fused to a protein. Commonly used and commercially available epitopetags include sequences derived from, e.g., influenza virus hemagglutinin(HA), Simian Virus 5 (V5), polyhistidine (6xHis), c-myc, lacZ, GST, andthe like. Antibodies specific to these epitope tags are generallycommercially available. Thus, the expressed reporter can be detectedusing an epitope-specific antibody in an immunoassay.

In another embodiment, the reporter is selected such that it can bedetected by a color-based assay. Examples of such reporters include,e.g., the lacZ protein (β-galactosidase), the green fluorescent protein(GFP), which can be detected by fluorescence assay and sorted byflow-activated cell sorting (FACS) (See Cubitt et al., Trends Biochem.Sci., 20:448-455 (1995)), secreted alkaline phosphatase, horseradishperoxidase, the blue fluorescent protein (BFP), and luciferasephotoproteins such as aequorin, obelin, mnemiopsin, and berovin (SeeU.S. Pat. No. 6,087,476, which is incorporated herein by reference).

Alternatively, an auxotrophic factor is used as a reporter in a hoststrain deficient in the auxotrophic factor. Thus, suitable auxotrophicreporter genes include, but are not limited to, URA3, HIS3, TRP1, LEU2,LYS2, ADE2, and the like. For example, yeast cells containing a mutantURA3 gene can be used as host cells (Ura⁻ phenotype). Such cells lackURA3-encoded functional orotidine-5′-phosphate decarboxylase, an enzymerequired by yeast cells for the biosynthesis of uracil. As a result, thecells are unable to grow on a medium lacking uracil. However, wild-typeorotidine-5′-phosphate decarboxylase catalyzes the conversion of anon-toxic compound 5-fluoroorotic acid (5-FOA) to a toxic product,5-fluorouracil. Thus, yeast cells containing a wild-type URA3 gene aresensitive to 5-FOA and cannot grow on a medium containing 5-FOA.Therefore, when the interaction between the interacting protein membersin the fusion proteins results in the expression of activeorotidine-5′-phosphate decarboxylase, the Ura⁻ (Foa^(R)) yeast cellswill be able to grow on a uracil deficient medium (SC-Ura plates).However, such cells will not survive on a medium containing 5-FOA. Thus,protein-protein interactions can be detected based on cell growth.

Additionally, antibiotic resistance reporters can also be employed in asimilar manner. In this respect, host cells sensitive to a particularantibiotic are used. Antibiotics resistance reporters include, forexample, the chloramphenicol acetyl transferase (CAT) gene and thekan^(R) gene, which confer resistance to G418 in eukaryotes, andkanamycin in prokaryotes, respectively.

5.3.13. Screening Assays for Interaction Antagonists

The screening assays of the present invention are useful in identifyingcompounds capable of interfering with or disrupting or dissociatingprotein-protein interactions between VAP-A, or a mutant form thereof,and a protein selected from the group consisting of PPP1R3 and GTR3, ora mutant form thereof. For example, VAP-A, or a mutant form thereof, andits interacting partners, or mutant forms thereof, are believed to playa role in docking and fusion of membrane vesicles to target organellemembranes and cellular uptake of glucose, and thus are involved indiabetes, obesity, ischemia, and insulin resistance. It may be possibleto ameliorate or alleviate the diseases or disorders in a patient byinterfering with or dissociating normal interactions between VAP-A andone of PPP1R3 and GTR3. Alternatively, if the disease or disorder isassociated with increased expression of VAP-A and/or one of theVAP-A-interacting proteins in accordance with the present invention,then the disease may be treated or prevented by weakening ordissociating the interaction between VAP-A and the VAP-A-interactingprotein in patients. In addition, if a disease or disorder is associatedwith mutant forms of VAP-A and/or one of the VAP-A-interacting proteinsthat lead to strengthened protein-protein interaction therebetween, thenthe disease or disorder may be treated with a compound that weakens orinterferes with the interaction between the mutant form of VAP-A and/orthe VAP-A-interacting protein(s).

In a screening assay for an interaction antagonist, VAP-A (or ahomologue, fragment or derivative thereof), or a mutant form of VAP-A(or a homologue, fragment or derivative thereof), and aVAP-A-interacting protein (or a homologue, fragment or derivativethereof), or a mutant form of a VAP-A-interacting protein (or ahomologue, fragment or derivative thereof), are used as test proteinsexpressed in the form of fusion proteins as described above for purposesof a two-hybrid assay. The fusion proteins are expressed in a host celland allowed to interact with each other in the presence of one or moretest compounds.

In a preferred embodiment, a counterselectable marker is used as areporter such that a detectable signal (e.g., appearance of color orfluorescence, or cell survival) is present only when the test compoundis capable of interfering with the interaction between the two testproteins. In this respect, the reporters used in various “reversetwo-hybrid systems” known in the art may be employed. Reverse two-hybridsystems are disclosed in, e.g., U.S. Pat. Nos. 5,525,490; 5,733,726;5,885,779; Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10315-10320(1996); and Vidal et al., Proc. Natl. Acad. Sci. USA, 93:10321-10326(1996), all of which are incorporated herein by reference.

Examples of suitable counterselectable reporters useful in a yeastsystem include the URA3 gene (encoding orotidine-5′-decarboxylase, whichconverts 5-fluroorotic acid (5-FOA) to the toxic metabolite5-fluorouracil), the CAN1 gene (encoding arginine permease, whichtransports toxic arginine analog canavanine into yeast cells), the GAL1gene (encoding galactokinase, which catalyzes the conversion of2-deoxygalactose to toxic 2-deoxygalactose-1-phosphate), the LYS2 gene(encoding α-aminoadipate reductase, which renders yeast cells unable togrow on a medium containing α-aminoadipate as the sole nitrogen source),the MET15 gene (encoding O-acetylhomoserine sulfhydrylase, which conferson yeast cells sensitivity to methyl mercury), and the CYH2 gene(encoding L29 ribosomal protein, which confers sensitivity tocycloheximide). In addition, any known cytotoxic agents includingcytotoxic proteins such as the diphtheria toxin (DTA) catalytic domaincan also be used as counterselectable reporters. See U.S. Pat.No.5,733,726. DTA causes the ADP-ribosylation of elongation factor-2 andthus inhibits protein synthesis and causes cell death. Other examples ofcytotoxic agents include ricin, Shiga toxin, and exotoxin A ofPseudomonas aeruginosa.

For example, when the URA3 gene is used as a counterselectable reportergene, yeast cells containing a mutant URA3 gene can be used as hostcells (Ura⁻Foa^(R) phenotype) for the in vivo assay. Such cells lackURA3-encoded-functional orotidine-5′-phosphate decarboxylase, an enzymerequired for the biosynthesis of uracil. As a result, the cells areunable to grow on media lacking uracil. However, because of the absenceof a wild-type orotidine-5′-phosphate decarboxylase, the yeast cellscannot convert non-toxic 5-fluoroorotic acid (5-FOA) to a toxic product,5-fluorouracil. Thus, such yeast cells are resistant to 5-FOA and cangrow on a medium containing 5-FOA. Therefore, for example, to screen fora compound capable of disrupting interactions between VAP-A (or ahomologue, fragment or derivative thereof), or a mutant form of VAP-A(or a homologue, fragment or derivative thereof), and PPP1R3 (or ahomologue, fragment or derivative thereof), or a mutant form of PPP1R3(or a homologue, fragment or derivative thereof), VAP-A (or ahonmologue, fragment or derivative thereof) can be expressed as a fusionprotein with a DNA-binding domain of a suitable transcription activatorwhile PPP1R3 (or a homologue, fragment or derivative thereof) isexpressed as a fusion protein with a transcription activation domain ofa suitable transcription activator. In the host strain, the reporterURA3 gene may be operably linked to a promoter specifically responsiveto the association of the transcription activation domain and theDNA-binding domain. After the fusion proteins are expressed in theUra⁻Foa^(R) yeast cells, an in vivo screening assay can be conducted inthe presence of a test compound with the yeast cells being cultured on amedium containing uracil and 5-FOA. If the test compound does notdisrupt the interaction between VAP-A and PPP1R3, active URA3 geneproduct, i.e., orotidine-5′-decarboxylase, which converts 5-FOA to toxic5-fluorouracil, is expressed. As a result, the yeast cells cannot grow.On the other hand, when the test compound disrupts the interactionbetween VAP-A and PPP1R3, no active orotidine-5′-decarboxylase isproduced in the host yeast cells. Consequently, the yeast cells willsurvive and grow on the 5-FOA-containing medium. Therefore, compoundscapable of interfering with or dissociating the interaction betweenVAP-A and PPP1R3 can thus be identified based on colony formation.

As will be apparent, the screening assay of the present invention can beapplied in a format appropriate for large-scale screening. For example,combinatorial technologies can be employed to construct combinatoriallibraries of small organic molecules or small peptides. See generally,e.g., Kenan et al., Trends Biochem. Sc., 19:57-64 (1994); Gallop et al.,J. Med. Chem., 37:1233-1251 (1994); Gordon et al., J. Med. Chem.,37:1385-1401 (1994); Ecker et al., Biotechnology, 13:351-360 (1995).Such combinatorial libraries of compounds can be applied to thescreening assay of the present invention to isolate specific modulatorsof particular protein-protein interactions. In the case of randompeptide libraries, the random peptides can be co-expressed with thefusion proteins of the present invention in host cells and assayed invivo. See e.g., Yang et al., Nucl. Acids Res., 23:1152-1156 (1995).Alternatively, they can be added to the culture medium for uptake by thehost cells.

Conveniently, yeast mating is used in an in vivo screening assay. Forexample, haploid cells of a-mating type expressing one fusion protein asdescribed above are mated with haploid cells of α-mating type expressingthe other fusion protein. Upon mating, the diploid cells are spread on asuitable medium to form a lawn. Drops of test compounds can be depositedonto different areas of the lawn. After culturing the lawn for anappropriate period of time, drops containing a compound capable ofmodulating the interaction between the particular test proteins in thefusion proteins can be identified by stimulation or inhibition of growthin the vicinity of the drops.

The screening assays of the present invention for identifying compoundscapable of modulating protein-protein interactions can also befine-tuned by various techniques to adjust the thresholds or sensitivityof the positive and negative selections. Mutations can be introducedinto the reporter proteins to adjust their activities. The uptake oftest compounds by the host cells can also be adjusted. For example,yeast high uptake mutants such as the erg6 mutant strains can facilitateyeast uptake of the test compounds. See Gaber et al., Mol. Cell. Biol.,9:3447-3456 (1989). Likewise, the uptake of the selection compounds suchas 5-FOA, 2-deoxygalactose, cycloheximide, α-aminoadipate, and the likecan also be fine-tuned.

5.3.1.4. Screening Assays for Interaction Agonists

The screening assays of the present invention can also be used inidentifying compounds that trigger or initiate, enhance or stabilizeprotein-protein interactions between VAP-A, or a mutant form thereof,and a protein selected from the group consisting of PPP1R3 and GTR3, ora mutant thereof. For example, if a disease or disorder is associatedwith decreased expression of VAP-A and/or a member of selected from thegroup of PPP1R3 and GTR3, then the disease or disorder may be treated orprevented by strengthening or stabilizing the interaction between VAP-Aand the VAP-A-interacting member in patients. Alternatively, if adisease or disorder is associated with mutant forms of VAP-A and/ormutant forms of a VAP-A-interacting protein that lead to weakened orabolished protein-protein interaction therebetween, then the disease ordisorder may be treated with a compound that initiates or stabilizes theinteraction between the mutant forms of VAP-A and/or the mutant forms ofVAP-A-interacting. protein(s).

Thus, a screening assay can be performed in the same manner as describedabove, except that a positively selectable marker is used. For example,VAP-A (or a homologue, fragment, or derivative thereof), or a mutantform of VAP-A (or a homologue, fragment, or derivative thereof), and aprotein selected from the group consisting of FPP1R3 and GTR3 (or ahomologue, fragment, or derivative thereof), or a mutant form of aprotein selected from the group consisting of PPP1R3 and GTR3 (or ahomologue, fragment, or derivative thereof), are used as test proteinsexpressed in the form of fusion proteins as described above for purposesof a two-hybrid assay. The fusion proteins are expressed in host cellsand are allowed to interact with each other in the presence of one ormore test compounds.

A gene encoding a positively selectable marker such as the lacZ proteinmay be used as a reporter gene such that when a test compound enables orenhances the interaction between VAP-A (or a homologue, fragment, orderivative thereof), or a mutant form of VAP-A (or a homologue,fragment, or derivative thereof), and a protein selected from the groupconsisting of PPP1R3 and GTR3 (or a homologue, fragment, or derivativethereof), or a mutant form of a protein selected from the groupconsisting of PPP1R3 and GTR3 (or a homologue, fragment, or derivativethereof), the lacZ protein, i.e., β-galatosidase, is expressed. As aresult, the compound may be identified based on the appearance of a bluecolor when the host cells are cultured in a medium containing X-Gal.

Generally, a control assay is performed in which the above screeningassay is conducted in the absence of the test compound. The result isthen compared with that obtained in the presence of the test compound.

5.4. Optimization of the Identified Compounds

Once the test compounds are selected capable of modulating theinteraction between PPP1R3 and a protein selected from PPP1R3 and GTR3,or modulating PPP1R3, or PPP1R3 and GTR3, a data set including datadefining the identity or characteristics of the test compounds can begenerated. The data set may include information relating to theproperties of a selected test compound, e.g., chemical structure,chirality, molecular weight, melting point, etc. Alternatively, the dataset may simply include assigned identification numbers understood by theresearchers conducting the screening assay and/or researchers receivingthe data set as representing specific test compounds. The data orinformation can be cast in a transmittable form that can be communicatedor transmitted to other researchers, particularly researchers in adifferent country. Such a transmittable form can vary and can betangible or intangible. For example, the data set defining one or moreselected test compounds can be embodied in texts, tables, diagrams,molecular structures, photographs, charts, images or any other visualforms. The data or information can be recorded on a tangible media suchas paper or embodied in computer-readable forms (e.g., electronic,electromagnetic, optical or other signals). The data in acomputer-readable form can be stored in a computer usable storage medium(e.g., floppy disks, magnetic tapes, optical disks, and the like) ortransmitted directly through a communication infrastructure. Inparticular, the data embodied in electronic signals can be transmittedin the form of email or posted on a website on the Internet or Intranet.In addition, the information or data on a selected test compound canalso be recorded in an audio form and transmitted through any suitablemedia, e.g., analog or digital cable lines, fiber optic cables, etc.,via telephone, facsimile, wireless mobile phone, Internet phone and thelike.

Thus, the information and data on a test compound selected in ascreening assay described above or by virtual screening as discussedbelow can be produced anywhere in the world and transmitted to adifferent location. For example, when a screening assay is conductedoffshore, the information and data on a selected test compound can begenerated and cast in a transmittable form as described above. The dataand information in a transmittable form thus can be imported into theU.S. or transmitted to any other countries, where the data andinformation may be used in further testing the selected test compoundand/or in modifying and optimizing the selected test compound to developlead compounds for testing in clinical trials.

Compounds can also be selected based on structural models of the targetprotein or protein complex and/or test compounds. In addition, once aneffective compound is identified, structural analogs or mimetics thereofcan be produced based on rational drug design with the aim of improvingdrug efficacy and stability, and reducing side effects. Methods known inthe art for rational drug design can be used in the present invention.See, e.g., Hodgson et al., Bio/Technology, 9:19-21 (1991); U.S. Pat.Nos. 5,800,998 and 5,891,628, all of which are incorporated herein byreference. An example of rational drug design is the development of HIVprotease inhibitors. See Erickson et al., Science, 249:527-533 (1990).

In this respect, structural information on the target protein or proteincomplex is obtained. Preferably, atomic coordinates defining athree-dimensional structure of the target protein or protein complex canbe obtained. For example, each of the interacting pair can be expressedand purified. The purified interacting protein pairs are then allowed tointeract with each other in vitro under appropriate conditions.Optionally, the interacting protein complex can be stabilized bycrosslinking or other techniques. The interacting complex can be studiedusing various biophysical techniques including, e.g., X-raycrystallography, NMR, computer modeling, mass spectrometry, and thelike. Likewise, structural information can also be obtained from proteincomplexes formed by interacting proteins and a compound that initiatesor stabilizes the interaction of the proteins. Methods for obtainingsuch atomic coordinates by X-ray crystallography, NMR, and the like areknown in the art and the application thereof to the target protein orprotein complex of the present invention should be apparent to skilledpersons in the art of structural biology. See Smyth and Martin, Mol.Pathol., 53:8-14 (2000); Oakley and Wilce, Clin. Exp. Pharmacol.Physiol., 27(3):145-151 (2000); Ferentz and Wagner, Q. Rev. Biophys.,33:29-65 (2000); Hicks, Curr. Med. Chem., 8(6):627-650 (2001); andRoberts, Curr. Opin. Biotechnol., 10:42-47 (1999).

In addition, understanding of the interaction between the proteins ofinterest in the presence or absence of a modulator can also be derivedfrom mutagenesis analysis using yeast two-hybrid system or other methodsfor detection protcin-protein interaction. In this respect, variousmutations can be introduced into the interacting proteins and the effectof the mutations on protein-protein interaction examined by a suitablemethod such as the yeast two-hybrid system.

Various mutations including amino acid substitutions, deletions andinsertions can be introduced into a protein sequence using conventionalrecombinant DNA technologies. Generally, it is particularly desirable todecipher the protein binding sites. Thus, it is important that themutations introduced only affect protein-protein interaction and causeminimal structural disturbances. Mutations are preferably designed basedon knowledge of the three-dimensional structure of the interactingproteins. Preferably, mutations are introduced to alter charged aminoacids or hydrophobic amino acids exposed on the surface of the proteins,since ionic interactions and hydrophobic interactions are often involvedin protein-protein interactions. Alternatively, the “alanine scanningmutagenesis” technique is used. See Wells, et al., Methods Enzymol.,202:301-306 (1991); Bass et al., Proc. Natl. Acad. Sci. USA,88:4498-4502 (1991); Bennet et al., J. Biol. Chem., 266:5191-5201(1991); Diamond et al., J. Virol., 68:863-876 (1994). Using thistechnique, charged or hydrophobic amino acid residues of the interactingproteins are replaced by alanine, and the effect on the interactionbetween the proteins is analyzed using e.g., the yeast two-hybridsystem. For example, the entire protein sequence can be scanned in awindow of five amino acids. When two or more charged or hydrophobicamino acids appear in a window, the charged or hydrophobic amino acidsare changed to alanine using standard recombinant DNA techniques. Thethus mutated proteins are used as “test proteins” in the above-describedtwo-hybrid assays to examine the effect of the mutations onprotein-protein interaction. Preferably, the mutagenesis analysis isconducted both in the presence and in the absence of an identifiedmodulator compound. In this manner, the domains or residues of theproteins important to protein-protein interaction and/or the interactionbetween the modulator compound and the interacting proteins can beidentified.

Based on the information obtained, structural relationships between theinteracting proteins, as well as between the identified modulators andthe interacting proteins are elucidated. For example, for the identifiedmodulators (i.e., lead compounds), the three-dimensional structure andchemical moieties critical to their modulating effect on the interactingproteins are revealed. Using this information and various techniquesknow in the art of molecular modeling (i.e., simulated annealing),medicinal chemists can then design analog compounds that might be moreeffective modulators of the protein-protein interactions of the presentinvention. For example, the analog compounds might show more specific ortighter binding to their targets, and thereby might exhibit fewer sideeffects, or might have more desirable pharmacological characteristics(e.g., greater solubility).

In addition, if the lead compound is a peptide, it can also be analyzedby the alanine scanning technique and/or the two-hybrid assay todetermine the domains or residues of the peptide important to itsmodulating effect on particular protein-protein interactions. Thepeptide compound can be used as a lead molecule for rational design ofsmall organic molecules or peptide mimetics. See Huber et al., Curr.Med. Chem., 1:13-34 (1994).

The domains, residues or moieties critical to the modulating effect ofthe identified compound constitute the active region of the compoundknown as its “pharmacophore.” Once the pharmacophore has beenelucidated, a structural model can be established by a modeling processthat may incorporate data from NMR analysis, X-ray diffraction data,alanine scanning, spectroscopic techniques and the like. Varioustechniques including computational analysis (e.g., molecular modelingand simulated annealing), similarity mapping and the like can all beused in this modeling process. See e.g., Perry et al., in OSAR:Quantitative Structure-Activity Relationships in Drug Design,pp.189-193, Alan R. Liss, Inc., 1989; Rotivinen et al., ActaPharmaceutical Fennica, 97:159-166 (1988); Lewis et al., Proc. R. Soc.Lond., 236:125-140 (1989); McKinaly et al., Annu. Rev. Pharmacol.Toxiciol., 29:111-122 (1989). Commercial molecular modeling systemsavailable from Polygen Corporation, Waltham, Mass., include the CHARMmprogram, which performs energy minimization and molecular dynamicsfunctions, and QUANTA program which performs construction, graphicmodeling and analysis of molecular structure. Such programs allowinteractive construction, modification and visualization of molecules.Other computer modeling programs are also available from BioDesign, Inc.(Pasadena, Calif.), Hypercube, Inc. (Cambridge, Ontario), and Allelix,Inc. (Mississauga, Ontario, Canada).

A template can be formed based on the established model. Variouscompounds can then be designed by linking various chemical groups ormoieties to the template. Various moieties of the template can also bereplaced. In addition, in the case of a peptide lead compound, thepeptide or mimetics thereof can be cyclized, e.g., by linking theN-terminus and C-terminus together, to increase its stability. Theserationally designed compounds are further tested. In this manner,pharmacologically acceptable and stable compounds with improved efficacyand reduced side effect can be developed. The compounds identified inaccordance with the present invention can be incorporated into apharmaceutical formulation suitable for administration to an individual.

In addition, the structural models or atomic coordinates defining athree-dimensional structure of the target protein or protein complex canalso be used in virtual screen to select compounds capable of modulatingthe target protein or protein complex. Various methods of computer-basedvirtual screen using atomic coordinates are generally known in the art.For example, U.S. Pat. No. 5,798,247 (which is incorporated herein byreference) discloses a method of identifying a compound (specifically,an interleukin converting enzyme inhibitor) by determining bindinginteractions between an organic compound and binding sites of a bindingcavity within the target protein. The binding sites are defined byatomic coordinates.

The compounds designed or selected based on rational drug design orvirtual screen can be tested for their ability to modulate (interferewith or strengthen) the interaction between the interacting partnerswithin the protein complexes of the present invention. In addition, thecompounds can also be further tested for their ability to modulate(inhibit or enhance) cellular functions such as docking and fusion ofmembrane vesicles to target organelle membranes and cellular uptake ofglucose in cells as well as their effectiveness in treating diseasessuch as diabetes, obesity, ischemia, and insulin resistance.

6. Therapeutic Applications

As described above, the interactions between VAP-A and theVAP-A-interacting proteins suggest that these proteins and/or theprotein complexes formed by them may be involved in common biologicalprocesses and disease pathways. The protein complexes may mediate thefunctions of VAP-A and the VAP-A-interacting proteins in the biologicalprocesses or disease pathways. Thus, one may modulate such biologicalprocesses or treat diseases by modulating the functions and activitiesof VAP-A, a VAP-A-interacting protein, and/or a protein complexcomprising some combination of these proteins. As used herein,modulating VAP-A, a VAP-A-interacting protein, or a protein complexcomprising some combination of these proteins means altering (enhancingor reducing) the concentrations or activities of the proteins or proteincomplexes, e.g., increasing the concentrations of VAP-A, aVAP-A-interacting protein or a protein complex comprising somecombination of these proteins, enhancing or reducing their biologicalactivities, increasing or decreasing their stability, altering theiraffinity or specificity to certain other biological molecules, etc. Forexample, a VAP-A-containing protein complex of the present invention orits members thereof may be involved in docking and fusion of membranevesicles to target organelle membranes and cellular uptake of glucose.Thus, assays such as those described in Section 4 may be used indetermining the effect of an aberration in a particular VAP-A-containingcomplex or an interacting member thereof on docking and fusion ofmembrane vesicles to target organelle membranes add cellular uptake ofglucose. In addition, it is also possible to determine, using the sameassay methods, the presence or absence of an association between aVAP-A-containing complex or an interacting member thereof and aphysiological disorder or disease such as diabetes, obesity, ischemia,and insulin resistance or predisposition to a physiological disorder ordisease.

Once such associations are established, the diagnostic methods asdescribed in Section 4 can be used in diagnosing the disease ordisorder, or a patient's predisposition to it. In addition, various invitro and in vivo assays may be employed to test the therapeutic orprophylactic efficacies of the various therapeutic approaches describedin Sections 6.2 and 6.3 that are aimed at modulating the functions andactivities of a particular VAP-A-containing complex of the presentinvention, or an interacting member thereof. Similar assays can also beused to test whether the therapeutic approaches described in Sections6.2 and 6.3 result in the modulation of docking and fusion of membranevesicles to target organelle membranes and cellular uptake of glucose.The cell model or transgenic animal model described in Section 7 may beemployed in the in vitro and in vivo assays.

In accordance with this aspect of the present invention, methods areprovided for modulating (promoting or inhibiting) a VAP-A-containingprotein complex or interacting member thereof. The human cells can be inin vitro cell or tissue cultures. The methods are also applicable tohuman cells in a patient.

In one embodiment, the concentration of a VAP-A-containing proteincomplex of the present invention is reduced in the cells. Variousmethods can be employed to reduce the concentration of the proteincomplex. The protein complex concentration can be reduced by interferingwith the interactions between the interacting members. For example,compounds capable of interfering with interactions between VAP-A and aprotein selected from the group of PPP1R3 and GTR3 can be administeredto the cells in vitro or in vivo in a patient. Such compounds can becompounds capable of binding VAP-A or the protein selected from PPP1R3and GTR3. They can also be antibodies immunoreactive with the VAP-A orthe protein selected from PPP1R3 and GTR3. Also, the compounds can besmall peptides derived from the a VAP-A-interacting protein or mimeticsthereof capable of binding VAP-A, or small peptides derived from VAP-Aprotein or mimetics thereof capable of binding a protein selected fromPPP1R3 and GTR3.

In another embodiment, the method of modulating the protein complexincludes inhibiting the expression of VAP-A protein and/or aVAP-A-interacting protein. The inhibition can be at the transcriptional,translational, or post-translational level. For example, antisensecompounds and ribozyme compounds can be administered to human cells incultures or in human bodies. In addition, RNA interference technologiesmay also be employed to administer to cells double-stranded RNA or RNAhairpins capable of “knocking down” the expression of VAP-A proteinand/or a VAP-A-interacting protein.

In the various embodiments described above, preferably theconcentrations or activities of both VAP-A protein and aVAP-A-interacting protein are reduced or inhibited.

In yet another embodiment, an antibody selectively immunoreactive with aprotein complex having VAP-A interacting with a protein selected fromPPP1R3 and GTR3 is administered to cells in vitro or in human bodies toinhibit the protein complex activities and/or reduce the concentrationof the protein complex in the cells or patient.

6.1. Applicable Diseases

The methods for modulating the functions and activities of aVAP-A-containing protein complex of the present invention, or aninteracting member thereof, may be employed to modulate docking andfusion of membrane vesicles to target organelle membranes and cellularuptake of glucose. In addition, the methods may also be used in thetreatment or prevention of diseases and disorders such as diabetes,obesity, ischemia, and insulin resistance.

6.2. Inhibiting Protein Complex or Interacting Protein Members Thereof

In one aspect of the present invention, methods are provided forreducing in cells or tissue the concentration and/or activity of aprotein complex identified in accordance with the present invention thatcomprises VAP-A and one or more members of the group PPP1R3 and GTR3. Inaddition, methods are also provided for reducing in cells or tissue theconcentration and/or activity of a VAP-A-interacting protein selectedfrom the group PPP1R3 and GTR3. By reducing the concentration of proteincomplex and/or the VAP-A-interacting protein concentration(s) and/orinhibiting the functional activities of the protein complex and/or theVAP-A-interacting protein(s), the diseases involving such proteincomplex or YAP-A-interacting protein(s) may be treated or prevented.

6.2.1. Antibody Therapy

In one embodiment, an antibody may be administered to cells or tissue invitro or to patients. The antibody administered may be immunoreactivewith VAP-A or a member of the group PPP1R3 and GTR3, or proteincomplexes comprising VAP-A and a member, or members, of the groupPPP1R3and GTR3. Suitable antibodies may be monoclonal or polyclonal thatfall within any antibody class, e.g., IgG, IgM, IgA, etc. The antibodysuitable for this invention may also take a form of various antibodyfragments including, but not limited to, Fab and F(ab′)₂, single-chainfragments (scFv), and the like. In another embodiment, an antibodyselectively immunoreactive with the protein complex formed from VAP-Aand one or more VAP-A-interacting protein, or proteins, in accordancewith the present invention is administered to cells or tissue in vitroor in a patient. In yet another embodiment, an antibody specific to aVAP-A-interacting protein selected from the group PPP1R3 and GTR3 isadministered to cells or tissue in vitro or in a patient. Methods formaking the antibodies of the present invention should be apparent to aperson of skill in the art, especially in view of the discussions inSection 3 above. The antibodies can be administered in any suitable formand route as described in Section 8 below. Preferably, the antibodiesare administered in a pharmaceutical composition together with apharmaceutically acceptable carrier.

Alternatively, the antibodies may be delivered by a gene-therapyapproach. That is, nucleic acids encoding the antibodies, particularlysingle-chain fragments (scFv), may be introduced into cells or tissue invitro or in a patient such that desirable antibodies may be producedrecombinantly in vivo from the nucleic acids. For this purpose, thenucleic acids with appropriate transcriptional and translationregulatory sequences can be directly administered into the patient.Alternatively, the nucleic acids can be incorporated into a suitablevector as described in Sections 2.2 and 5.3.1.1 and delivered into cellsor tissue in vitro or in a patient along with the vector. The expressionvector containing the nucleic acids can be administered directly tocells or tissue in vitro or in a patient. It can also be introduced intocells, preferably cells derived from a patient to be treated, andsubsequently delivered into the patient by cell transplantation. SeeSection 6.3.2 below.

6.2.2. Antisense Therapy

In another embodiment, antisense compounds specific to nucleic acidsencoding one or more interacting protein members of a protein complexidentified in the present invention are administered to cells or tissuein vitro or in a patient to be therapeutically or prophylacticallytreated. The antisense compounds should specifically inhibit theexpression of the one or more interacting protein members. As is knownin the art, antisense drugs generally act by hybridizing to a particulartarget nucleic acid thus blocking gene expression. Methods for designingantisense compounds and using such compounds in treating diseases arewell known and well developed in the art. For example, the antisensedrug Vitravene® (fomivirsen), a 21-base long oligonucleotide, has beensuccessfully developed and marketed by Isis Pharmaceuticals, Inc. fortreating cytomegalovirus (CMV)-induced retinitis.

Any methods for designing and making antisense compounds may be used forpurpose of the present invention. See generally, Sanghvi et al., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993.Typically, antisense compounds are oligonucleotides designed based onthe nucleotide sequence of the mRNA or gene of one or more targetproteins, e.g., the interacting protein members of a particular proteincomplex of the present invention. In particular, antisense compounds canbe designed to specifically hybridize to a particular region of the genesequence or mRNA of one or more of the interacting protein membersto.modulate (increase or decrease), replication, transcription, ortranslation. As used herein, the term “specifically hybridize” orparaphrases thereof means a sufficient degree of complementarity orpairing between an antisense oligo and a target DNA or mRNA such thatstable and specific binding occurs therebetween. In particular, 100%complementary or pairing is not required. Specific hybridization takesplace when sufficient hybridization occurs between the antisensecompound and its intended target nucleic acids in the substantialabsence of non-specific binding of the antisense compound to non-targetsequences under predetermined conditions, e.g., for purposes of in vivotreatment, preferably under physiological conditions. Preferably,specific hybridization results in the interference with normalexpression of the target DNA or mRNA.

For example, antisense oligonucleotides can be designed to specificallyhybridize to target genes, in regions critical for regulation oftranscription; to pre-mRNAs, in regions critical for correct splicing ofnascent transcripts; and to mature mRNAs, in regions critical fortranslation initiation or mRNA stability and localization.

As is generally known in the art, commonly used oligonucleotides areoligomers or polymers of ribonucleotides or deoxyribonucleotides, thatare composed of a naturally-occurring nitrogenous base, a sugar (riboseor deoxyribose) and a phosphate group. In nature, the nucleotides arelinked together by phosphodiester bonds between the 3′ and 5′ positionsof neighboring sugar moieties. However, it is noted that the term“oligonucleotides” also encompasses various non-naturally occurringmimetics and derivatives, i.e., modified forms, of naturally occurringoligonucleotides as described below. Typically an antisense compound ofthe present invention is an oligonucleotide having from about 6 to about200, and preferably from about 8 to about 30 nucleoside bases.

The antisense compounds preferably contain modified backbones ornon-natural internucleoside linkages, including but not limited to,modified phosphorous-containing backbones and non-phosphorous backbonessuch as morpholino backbones; siloxane, sulfide, sulfoxide, sulfone,sulfonate, sulfonamide, and sulfamate backbones; formacetyl andthioformacetyl backbones; alkene-containing backbones; methyleneiminoand methylenehydrazino backbones; amide backbones, and the like.

Examples of modified phosphorous-containing backbones include, but arenot limited to phosphorothioates, phosphorodithioates, chiralphosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphotriesters, andboranophosphates and various salt forms thereof. See e.g., U.S. Pat.Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and5,625,050, each of which is herein incorporated by reference.

Examples of the non-phosphorous containing backbones described above aredisclosed in, e.g., U.S. Pat. Nos. 5,034,506; 5,185,444; 5,214,134;5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,677,437; and 5,677,439, each of which is herein incorporated byreference.

Another useful modified oligonucleotide is peptide nucleic acid (PNA),in which the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, e.g., an aminoethylglycine backbone. See U.S.Pat. Nos. 5,539,082 and 5,714,331; and Nielsen et al., Science, 254,1497-1500 (1991), all of which are incorporated herein by reference. PNAantisense compounds are resistant to RNase H digestion and thus exhibitlonger half-life. In addition, various modifications may be made in PNAbackbones to impart desirable drug profiles such as better stability,increased drug uptake, higher affinity to target nucleic acid, etc.

Alternatively, the antisense compounds are oligonucleotides containingmodified nucleosides, i.e., modified purine or pyrimidine bases, e.g.,5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 andO-substituted purines, and the like. See e.g., U.S. Pat. Nos. 3,687,808;4,845,205; 5,130,302; 5,175,273; 5,367,066; 5,432,272; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,587,469; 5,594,121; 5,596,091;5,681,941; and 5,750,692, each of which is incorporated herein byreference in its entirety.

In addition, oligonucleotides with substituted or modified sugarmoieties may also be used. For example, an antisense compound may haveone or more 2′-O-methoxyethyl sugar moieties. See e.g., U.S. Pat. Nos.4,981,957; 5,118,800; 5,319,080; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,567,811; 5,576,427; 5,591,722; 5,610,300; 5,627,05315,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of whichis herein incorporated by reference.

Other types of oligonucleotide modifications are also useful includinglinking an oligonucleotide to a lipid, phospholipid or cholesterolmoiety, cholic acid, thioether, aliphatic chain, polyamine, polyethyleneglycol (PEG), or a protein or peptide. The modified oligonucleotides mayexhibit increased uptake into cells, and improved stability, i.e.,resistance to nuclease digestion and other biodegradations. See e.g.,U.S. Pat. No. 4,522,811; Burnham, Am. J. Hosp. Pharm., 15:210-218(1994).

Antisense compounds can be synthesized using any suitable methods knownin the art. In fact, antisense compounds may be custom made bycommercial suppliers. Alternatively, antisense compounds may be preparedusing DNA synthesizers available commercially from various vendors,e.g., Applied Biosystems Group of Norwalk, Conn.

The antisense compounds can be formulated into a pharmaceuticalcomposition with suitable carriers and administered into cells or tissuein vitro or in a patient using any suitable route of administration.Alternatively, the antisense compounds may also be used in a“gene-therapy” approach. That is, the oligonucleotide is subcloned intoa suitable vector and transformed into human cells. The antisenseoligonucleotide is then produced in vivo through transcription. Methodsfor gene therapy are disclosed in Section 6.3.2 below.

6.23. Ribozyme Therapy

In another embodiment, an enzymatic RNA or ribozyme is designed totarget the nucleic acids encoding one or more of the interacting proteinmembers of the protein complex of the present invention. Ribozymes areRNA molecules possessing enzymatic activity. One class of ribozymes iscapable of repeatedly cleaving other separate RNA molecules into two ormore pieces in a nucleotide base sequence specific manner. See Kim etal., Proc. Natl. Acad. of Sci. USA, 84:8788 (1987); Haseloff andGerlach, Nature, 334:585 (1988); and Jefferies et al., Nucleic AcidRes., 17:1371 (1989). Such ribozymes typically have two functionaldomains: a catalytic domain and a binding sequence that guides thebinding of ribozymes to a target RNA through complementary base-pairing.Once a specifically-designed ribozyme is bound to a target mRNA, itenzymatically cleaves the target mRNA, typically reducing its stabilityand destroying its ability to direct translation of an encoded protein.After a ribozyme has cleaved its RNA target, it is released from thattarget RNA and thereafter can bind and cleave another target. That is, asingle ribozyme molecule can repeatedly bind and cleave new targets.Therefore, one advantage of ribozyme treatment is that a lower amount ofexogenous RNA is required as compared to conventional antisensetherapies. In addition, ribozymes exhibit less affinity to mRNA targetsthan DNA-based antisense oligonucleotides, and therefore are less proneto bind to wrong targets.

In accordance with the present invention, a ribozyme may target anyportion of the mRNA of one or more interacting protein members includingVAP-A, and PPP1R3 and GTR3. Methods for selecting a ribozyme targetsequence and designing and making ribozymes are generally known in theart. See e.g., U.S. Pat. Nos. 4,987,071; 5,496,698; 5,525,468;5,631,359; 5,646,020; 5,672,511; and 6,140,491, each of which isincorporated herein by reference in its entirety. For example, suitableribozymes may be designed in various configurations such as hammerheadmotifs, hairpin motifs, hepatitis delta virus motifs, group I intronmotifs, or RNase P RNA motifs. See e.g., U.S. Pat. Nos. 4,987,071;5,496,698; 5,525,468; 5,631,359; 5,646,020; 5,672,511; and 6,140,491;Rossi et al., AIDS Res. Human Retroviruses 8:183 (1992); Hampel andTritz, Biochemistry 28:4929 (1989); Hampel et al., Nucleic Acids Res.,18:299 (1990); Perrotta and Been, Biochemistry 31:16 (1992); andGuerrier-Takada et al., Cell, 35:849 (1983).

Ribozymes can be synthesized by the same methods used for normal RNAsynthesis. For example, such methods are disclosed in Usman et al., J.Am. Chem. Soc., 109:7845-7854 (1987) and Scaringe et al., Nucleic AcidsRes., 18:5433-5441 (1990). Modified ribozymes may be synthesized by themethods disclosed in, e.g., U.S. Pat. No. 5,652,094; InternationalPublication Nos. WO 91/03162; WO 92/07065 and WO 93/15187; European Pat.Application No. 92110298.4; Perrault et al., Nature, 344:565 (1990);Pieken et al., Science, 253:314 (1991); and Usman and Cedergren, Trendsin Biochem. Sci., 17:334 (1992).

Ribozymes of the present invention may be administered to cells by anyknown methods, e.g., disclosed in International Publication No. WO94/02595. For example, they can be administered directly to cells ortissue in vitro or in a patient through any suitable route, e.g.,intravenous injection. Alternatively, they may be delivered encapsulatedin liposomes, by iontophoresis, or by incorporation into other vehiclessuch as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. In addition, they may also be delivered bygene therapy approach, using a DNA vector from which the ribozyme RNAcan be transcribed directly. Gene therapy methods are disclosed indetail below in Section 6.3.2.

6.2.4. Other Methods

The in-patient concentrations and activities of the protein complexesand interacting proteins of the present invention may also be altered byother methods. For example, compounds identified in accordance with themethods described in Section 5 that are capable of interfering with ordissociating protein-protein interactions between the interactingprotein members of a protein complex may be administered to cells ortissue in vitro or in a patient. Compounds identified in in vitrobinding assays described in Section 5.2 that bind to theVAP-A-containing protein complex or the interacting members thereof mayalso be used in the treatment. Compounds identified in in vitro bindingassays described in Section 5.2 that bind to the VAP-A-containingprotein complex, or the interacting members thereof, may also be used inthe treatment.

In addition, potentially useful agents also include incomplete proteins,i.e., fragments of the interacting protein members that are capable ofbinding to their respective binding partners in a protein complex butare defective with respect to their normal cellular functions. Forexample, binding domains of the interacting member proteins of a proteincomplex may be used as competitive inhibitors of the activities of theprotein complex. As will be apparent to skilled artisans, derivatives orhomologues of the binding domains may also be used. Binding domains canbe easily identified using molecular biology techniques, e.g.,mutagenesis in combination with yeast two-hybrid assays. Preferably, theprotein fragment used is a fragment of an interacting protein memberhaving a length of less than 90%, 80%, more preferably less than 75%,65%, 50%, or less than 40% of the full length of the protein member. Inone embodiment, a VAP-A protein fragment is administered. In a specificembodiment, one or more of the interaction domains of VAP-A within theregions listed in Table 1 are administered to cells or tissue in vitro,or are administered to a patient in need of such treatment. For example,suitable protein fragments can include polypeptides having a contiguousspan of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20 or 25,preferably from 4 to 30, 40 or 50 amino acids or more of the sequence ofVAP-A that are capable of interacting with one or more proteins selectedfrom the group of PPP1R3 and GTR3. Also, suitable protein fragments canalso include peptides capable of binding one or more proteins selectedfrom the group of PPP1R3 and GTR3 and having an amino acid sequence offrom 4 to 30 amino acids that is at least 75%, 80%, 82%, 85%, 87%, 90%,95% or more identical to a contiguous span of amino acids of VAP-A ofthe same length. Alternatively, a polypeptide capable of interactingwith VAP-A and having a contiguous span of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 18, 20 or 25, preferably from 4 to 30, 40 or 50 or moreamino acids of the amino acid sequence of a protein selected from thegroup of PPP1R3 and GTR3 may be administered. Also, other examples ofsuitable compounds include a peptide capable of binding VAP-A and havingan amino acid sequence of from 4 to 30, 40, 50 or more amino acids thatis at least 75%, 80%, 82%, 85%, 87%, 90%, 92%, 95% or more identical toa contiguous span of amino acids of the same length from a proteinselected from the group of PPP1R3 and GTR3. In addition, theadministered compounds can be an antibody or antibody fragment,preferably single-chain antibody immunoreactive with VAP-A or a proteinselected from the group of PPP1R3 and GTR3, or a protein complex of thepresent invention.

The protein fragments suitable as competitive inhibitors can bedelivered into cells by direct cell internalization, receptor mediatedendocytosis, or via a “transporter.” It is noted that when the targetproteins or protein complexes to be modulated reside inside cells, thecompound administered to cells in vitro or in vivo in the method of thepresent invention preferably is delivered into the cells in order toachieve optimal results. Thus, preferably, the compound to be deliveredis associated with a transporter capable of increasing the uptake of thecompound by cells harboring the target protein or protein complex. Asused herein, the term “transporter” refers to an entity (e.g., acompound or a composition or a physical structure formed from multiplecopies of a compound or multiple different compounds) that is capable offacilitating the uptake of a compound of the present invention by animalcells, particularly human cells. Typically, the cell uptake of acompound of the present invention in the presence of a “transporter” isat least 20% higher, preferably at least 40%, 50%, 75%, and morepreferably at least 100% higher than the cell uptake of the compound inthe absence of the “transporter.”

Many molecules and structures known in the art can be used as“transporters.” In one embodiment, a penetratin is used as atransporter. For example, the homeodomain of Antennapedia, a Drosophilatranscription factor, can be used as a transporter to deliver a compoundof the present invention. Indeed, any suitable member of the penetratinclass of peptides can be used to carry a compound of the presentinvention into cells. Penetratins are disclosed in, e.g., Derossi etal., Trends Cell Biol., 8:84-87 (1998), which is incorporated herein byreference. Penetratins transport molecules attached thereto acrosscytoplasmic membranes or nuclear membranes efficiently, in areceptor-independent, energy-independent, and cell type-independentmanner. Methods for using a penetratin as a carrier to deliveroligonucleotides and polypeptides are also disclosed in U.S. Pat. No.6,080,724; Pooga et al., Nat. Biotech., 16:857 (1998); and Schutze etal., J. Immunol., 157:650 (1996), all of which are incorporated hereinby reference. U.S. Pat. No. 6,080,724 defines the minimal requirementsfor a penetratin peptide as a peptide of 16 amino acids with 6 to 10 ofwhich being hydrophobic. The amino acid at position 6 counting fromeither the N- or C-terminus is tryptophan, while the amino acids atpositions 3 and 5 counting from either the N- or C-terminus are not bothvaline. Preferably, the helix 3 of the homeodomain of DrosophilaAntennapedia is used as a transporter. More preferably, a peptide havinga sequence of amino acid residues 43-58 of the homeodomain Antp isemployed as a transporter. In addition, other naturally occurringhomologs of the helix 3 of the homeodomain of Drosophila Antennapediacan be used. For example, homeodomains of Fushi-tarazu and Engrailedhave been shown to be capable of transporting peptides into cells. SeeHan et al., Mol. Cells, 10:728-32 (2000). As used herein, the term“penetratin” also encompasses peptoid analogs of the penetratinpeptides. Typically, the penetratin peptides and peptoid analogs thereofare covalently linked to a compound to be delivered into cells thusincreasing the cellular uptake of the compound.

In another embodiment, the HIV-1 tat protein or a derivative thereof isused as a “transporter” covalently linked to a compound according to thepresent invention. The use of HIV-1 tat protein and derivatives thereofto deliver macromolecules into cells has been known in the art. SeeGreen and Loewenstein, Cell, 55:1179 (1988); Frankel and Pabo, Cell,55:1189 (1988); Vives et al., J. Biol. Chem., 272:16010-16017 (1997);Schwarze et al., Science, 285:1569-1572 (1999). It is known that thesequence responsible for cellular uptake consists of the highly basicregion, amino acid residues 49-57. See e.g., Vives et al., J. Biol.Chem., 272:16010-16017 (1997); Wender et al., Proc. Nat'l Acad. Sci.USA, 97:13003-13008 (2000). The basic domain is believed to target thelipid bilayer component of cell membranes. It causes a covalently linkedprotein or nucleic acid to cross cell membrane rapidly in a celltype-independent manner. Proteins ranging in size from 15 to 120 kD havebeen delivered with this technology into a variety of cell types both invitro and in vivo. See Schwarze et al., Science, 285:1569-1572 (1999).Any HIV tat-derived peptides or peptoid analogs thereof capable oftransporting macromolecules such as peptides can be used for purposes ofthe present invention. For example, any native tat peptides having thehighly basic region, amino acid residues 49-57 can be used as atransporter by covalently linking it to the compound to be delivered. Inaddition, various analogs of the tat peptide of amino acid residues49-57 can also be useful transporters for purposes of this invention.Examples of various such analogs are disclosed in Wender et al., Proc.Nat'l. Acad. Sci. USA, 97:13003-13008 (2000) (which is incorporatedherein by reference) including, e.g., d-Tat₄₉₋₅₇, retro-inverso isomersof l- or d-Tat₄₉₋₅₇ (i.e., l-Tat₅₇₋₄₉ and d-Tat₅₇₋₄₉), L-arginineoligomers, D-arginine oligomers, L-lysine oligomers, D-lysine oligomers,L-histine oligomers, D-histine oligomers, L-ornithine oligomers,D-ornithine oligomers, and various homologues, derivatives (e.g.,modified forms with conjugates linked to the small peptides) and peptoidanalogs thereof.

Other useful transporters known in the art include, but are not limitedto, short peptide sequences derived from fibroblast growth factor (SeeLin et al., J. Biol. Chem., 270:14255-14258 (1998)), Galparan (See Poogaet al., FASEB J. 12:67-77 (1998)), and HSV-1 structural protein VP22(See Elliott and O'Hare, Cell, 88:223-233 (1997)).

As the above-described various transporters are generally peptides,fusion proteins can be conveniently made by recombinant expression tocontain a transporter peptide covalently linked by a peptide bond to acompetitive protein fragment. Alternatively, conventional methods can beused to chemically synthesize a transporter peptide or a peptide of thepresent invention or both.

The hybrid peptide can be administered to cells or tissue in vitro or toa patient in a suitable pharmaceutical composition as provided inSection 8.

In addition to peptide-based transporters, various other types oftransporters can also be used, including but not limited to cationicliposomes (see Rui et al., J. Am. Chem. Soc., 120:11213-11218 (1998)),dendrimers (Kono et al., Bioconjugate Chem., 10:1115-1121 (1999)),siderophores (Ghosh et al., Chem. Biol., 3:1011-1019 (1996)), etc. In aspecific embodiment, the compound according to the present invention isencapsulated into liposomes for delivery into cells.

Additionally, when a compound according to the present invention is apeptide, it can be administered to cells by a gene therapy method. Thatis, a nucleic acid encoding the peptide can be administered to in vitrocells or to cells in vivo in a human or animal body. Any suitable genetherapy methods may be used for purposes of the present invention.Various gene therapy methods are well known in the art and are describedin Section 6.3.2. below. Successes in gene therapy have been reportedrecently. See e.g., Kay et al., Nature Genet., 24:257-61 (2000);Cavazzana-Calvo et al., Science, 288:669 (2000); and Blaese et al.,Science, 270: 475 (1995); Kantoff, et al., J. Exp. Med., 166:219 (1987).

In yet another embodiment, the gene therapy methods discussed in Section6.3.2 below are used to “knock out” the gene encoding an interactingprotein member of a protein complex, or to reduce the gene expressionlevel. For example, the gene may be replaced with a different genesequence or a non-functional sequence or simply deleted by homologousrecombination. In another gene therapy embodiment, the method disclosedin U.S. Pat. No. 5,641,670, which is incorporated herein by reference,may be used to reduce the expression of the genes for the interactingprotein members. Essentially, an exogenous DNA having at least aregulatory sequence, an exon and a splice donor site can be introducedinto an endogenous gene encoding an interacting protein member byhomologous recombination such that the regulatory sequence, the exon andthe splice donor site present in the DNA construct become operativelylinked to the endogenous gene. As a result, the expression of theendogenous gene is controlled by the newly introduced exogenousregulatory sequence. Therefore, when the exogenous regulatory sequenceis a strong gene expression repressor, the expression of the endogenousgene encoding the interacting protein member is reduced or blocked. SeeU.S. Pat. No. 5,641,670.

6.3. Activation of Protein Complex or Interacting Protein MembersThereof

The present invention also provides methods for increasing in cells ortissue in vitro or in a patient the concentration and/or activity of aprotein complex, or of an individual protein member thereof, identifiedin accordance with the present invention. Such methods can beparticularly useful in instances where a reduced concentration and/oractivity of a protein complex, or a protein member thereof, isassociated with a particular disease or disorder to be treated, or wherean increased concentration and/or activity of a protein complex, or aprotein member thereof, would be beneficial to the improvement of acellular function or disease state. By increasing the concentration ofthe protein complex, or a protein member thereof, and/or stimulating thefunctional activities of the protein complex or a protein memberthereof, the disease or disorder may be treated or prevented.

6.3.1. Administration of Protein Complex or Protein Members Thereof

Where the concentration or activity of a particular VAP-A-containingprotein complex, or VAP-A itself, or a VAP-A-interacting protein of thepresent invention, in cells or tissue in vitro or in a patient isdetermined to be low or is desired to be increased, the protein complex,or VAP-A, or the VAP-A-interacting protein may be administered directlyto the patient to increase the concentration and/or activity of theprotein complex, VAP-A, or the VAP-A-interacting protein. For thispurpose, protein complexes prepared by any one of the methods describedin Section 2.2 may be administered to the patient, preferably in apharmaceutical composition as described below. Alternatively, one ormore individual interacting protein members of the protein complex mayalso be administered to the patient in need of treatment. For example,one or more proteins such as VAP-A, PPP1R3 and GTR3 may be given tocells or tissue in vitro or to a patient. Proteins isolated or purifiedfrom normal individuals or recombinantly produced can all be used inthis respect. Preferably, two or more interacting protein members of aprotein complex are administered. The proteins or protein complexes maybe administered to a patient needing treatment using any of the methodsdescribed in Section 8.

6.3.2. Gene Therapy

In another embodiment, the concentration and/or activity of a particularVAP-A-containing protein complex or VAP-A, or a known VAP-A-interactingprotein (selected from the group including PPP1R3 and GTR3) is increasedor restored in patients, tissue or cells by a gene therapy approach. Forexample, nucleic acids encoding one or more protein members of aVAP-A-containing protein complex of the present invention, or portionsor fragments thereof are introduced into patients, tissue, or cells suchthat the protein(s) are expressed from the introduced nucleic acids. Forthese purposes, nucleic acids encoding one or more of VAP-A, PPP1R3 andGTR3, or fragments, homologues or derivatives thereof can be used in thegene therapy in accordance with the present invention. For example, if adisease-causing mutation exists in one of the protein members in cellsor tissue in vitro or in a patient, then a nucleic acid encoding awild-type protein can be introduced into tissue cells of the patient.The exogenous nucleic acid can be used to replace the correspondingendogenous defective gene by, e.g., homologous recombination. See U.S.Pat. No. 6,010,908, which is incorporated herein by reference.Alternatively, if the disease-causing mutation is a recessive mutation,the exogenous nucleic acid is simply used to express a wild-type proteinin addition to the endogenous mutant protein. In another approach, themethod disclosed in U.S. Pat. No. 6,077,705 may be employed in genetherapy. That is, the patient is administered both a nucleic acidconstruct encoding a ribozyme and a nucleic acid construct comprising aribozyme resistant gene encoding a wild type form of the gene product.As a result, undesirable expression of the endogenous gene is inhibitedand a desirable wild-type exogenous gene is introduced. In yet anotherembodiment, if the endogenous gene is of wild-type and the level ofexpression of the protein encoded thereby is desired to be increased,additional copies of wild-type exogenous genes may be introduced intothe patient by gene therapy, or alternatively, a gene activation methodsuch as that disclosed in U.S. Pat. No. 5,641,670 may be used.

Various gene therapy methods are well known in the art. Successes ingene therapy have been reported recently. See e.g., Kay et al., NatureGenet., 24:257-61 (2000); Cavazzana-Calvo et al., Science, 288:669(2000); and Blaese et al., Science, 270: 475 (1995); Kantoff, et al., J.Exp. Med. 166:219 (1987).

Any suitable gene therapy methods may be used for the purposes of thepresent invention. Generally, a nucleic acid encoding a desirableprotein (e.g., one selected from VAP-A, PPP1R3 and GTR3) is incorporatedinto a suitable expression vector and is operably linked to a promoterin the vector. Suitable promoters include but are not limited to viraltranscription promoters derived from adenovirus, simian virus 40 (SV40)(e.g., the early and late promoters of SV40), Rous sarcoma virus (RSV),and cytomegalovirus (CMV) (e.g., CMV immediate-early promoter), humanimmunodeficiency virus (HIV) (e.g., long terminal repeat (LTR)),vaccinia virus (e.g., 7.5K promoter), and herpes simplex virus (HSV)(e.g., thymidine kinase promoter). Where tissue-specific expression ofthe exogenous gene is desirable, tissue-specific promoters may beoperably linked to the exogenous gene. In addition, selection markersmay also be included in the vector for purposes of selecting, in vitro,those cells that contain the exogenous gene. Various selection markersknown in the art may be used including, but not limited to, e.g., genesconferring resistance to neomycin, hygromycin, zeocin, and the like.

In one embodiment, the exogenous nucleic acid (gene) is incorporatedinto a plasmid DNA vector. Many commercially available expressionvectors may be useful for the present invention, including, e.g., pCEP4,pcDNAI, pIND, pSecTag2, pVAX1, pcDNA3.1, and pBI-EGFP, and pDisplay.

Various viral vectors may also be used. Typically, in a viral vector,the viral genome is engineered to eliminate the disease-causingcapability of the virus, e.g., the ability to replicate in the hostcells. The exogenous nucleic acid to be introduced into cells or tissuein vitro or in a patient may be incorporated into the engineered viralgenome, e.g., by inserting it into a viral gene that is non-essential tothe viral infectivity. Viral vectors are convenient to use as they canbe easily introduced into cells, tissues and patients by way ofinfection. Once in the host cell, the recombinant virus typically isintegrated into the genome of the host cell. In rare instances, therecombinant virus may also replicate and remain as extrachromosomalelements.

A large number of retroviral vectors have been developed for genetherapy. These include vectors derived from oncoretroviruses (e.g.,MLV), lentiviruses (e.g., HIV and SIV) and other retroviruses. Forexample, gene therapy vectors have been developed based on murineleukemia virus (See, Cepko, et al., Cell, 37:1053-1062 (1984), Cone andMulligan, Proc. Natl. Acad. Sci. U.S.A., 81:6349-6353 (1984)), mousemammary tumor virus (See, Salmons et al., Biochem. Biophys. Res.Commun., 159:1191-1198 (1984)), gibbon ape leukemia virus (See, Milleret al., J. Virology, 65:2220-2224 (1991)), HIV, (See Shimada et al., J.Clin. Invest., 88:1043-1047 (1991)), and avian retroviruses (See Cossetet al., J. Virology, 64:1070-1078 (1990)). In addition, variousretroviral vectors are also described in U.S. Pat. Nos. 6,168,916;6,140,111; 6,096,534; 5,985,655; 5,911,983; 4,980,286; and 4,868,116,all of which are incorporated herein by reference.

Adeno-associated virus (AAV) vectors have been successfully tested inclinical trials. See e.g., Kay et al., Nature Genet. 24:257-61 (2000).AAV is a naturally occurring defective virus that requires other virusessuch as adenoviruses or herpes viruses as helper viruses. See Muzyczka,Curr. Top. Microbiol. Immun., 158:97 (1992). A recombinant AAV virususeful as a gene therapy vector is disclosed in U.S. Pat. No. 6,153,436,which is incorporated herein by reference.

Adenoviral vectors can also be useful for purposes of gene therapy inaccordance with the present invention. For example, U.S. Pat. No.6,001,816 discloses an adenoviral, which is used to deliver a leptingene intravenously to a mammal to treat obesity. Other recombinantadenoviral vectors may also be used, which include those disclosed inU.S. Pat. Nos. 6,171,855; 6,140,087; 6,063,622; 6,033,908; and5,932,210, and Rosenfeld et al., Science, 252:431434 (1991); andRosenfeld et al., Cell, 68:143-155 (1992).

Other useful viral vectors include recombinant hepatitis viral vectors(See, e.g., U.S. Pat. No. 5,981,274), and recombinant entomopox vectors(See, e.g., U.S. Pat. Nos. 5,721,352 and 5,753,258).

Other non-traditional vectors may also be used for purposes of thisinvention. For example, International Publication No. WO 94/18834discloses a method of delivering DNA into mammalian cells by conjugatingthe DNA to be delivered with a polyelectrolyte to form a complex. Thecomplex may be microinjected into or taken up by cells.

The exogenous gene fragment or plasmid DNA vector containing theexogenous gene may also be introduced into cells by way ofreceptor-mediated endocytosis. See e.g., U.S. Pat. No. 6,090,619; Wu andWu, J. Biol. Chem., 263:14621 (1988); Curiel et al., Proc. Natl. Acad.Sci. USA, 88:8850 (1991). For example, U.S. Pat. No. 6,083,741 disclosesintroducing an exogenous nucleic acid into mammalian cells byassociating the nucleic acid to a polycation moiety (e.g., poly-L-lysinehaving 3-100 lysine residues), which is itself coupled to an integrinreceptor binding moiety (e.g., a cyclic peptide having the sequenceArg-Gly-Asp).

Alternatively, the exogenous nucleic acid or vectors containing it canalso be delivered into cells via amphiphiles. See e.g., U.S. Pat. No.6,071,890. Typically, the exogenous nucleic acid or a vector containingthe nucleic acid forms a complex with the cationic amphiphile. Mammaliancells contacted with the complex can readily take it up.

The exogenous gene can be introduced into cells or tissue in vitro or ina patient for purposes of gene therapy by various methods known in theart. For example, the exogenous gene sequences alone or in a conjugatedor complex form described above, or incorporated into viral or DNAvectors, may be administered directly by injection into an appropriatetissue or organ of a patient. Alternatively, catheters or like devicesmay be used to deliver exogenous gene sequences, complexes, or vectorsinto a target organ or tissue. Suitable catheters are disclosed in,e.g., U.S. Pat. Nos. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and6,129,705, all of which are incorporated herein by reference.

In addition, the exogenous gene or vectors containing the gene can beintroduced into isolated cells using any known techniques such ascalcium phosphate precipitation, microinjection, lipofection,electroporation, biolystics, receptor-mediated endocytosis, and thelike. Cells expressing the exogenous gene may be selected andredelivered back to the patient by, e.g., injection or celltransplantation. The appropriate amount of cells delivered to a patientwill vary with patient conditions, and desired effect, which can bedetermined by a skilled artisan. See e.g., U.S. Pat. Nos. 6,054,288;6,048,524; and 6,048,729. Preferably, the cells used are autologous,i.e., cells obtained from the patient being treated.

6.3.3. Small Organic Compounds

Defective conditions or disorders in cells or tissue in vitro or in apatient associated with decreased concentration or activity of aVAP-A-containing protein complex, VAP-A, or a VAP-A-interacting proteinidentified in accordance with the present invention, can also beameliorated by administering to the patient a compound identified by themethods described in Sections 5.3.1.4, 5.2, and Section 5.4, which iscapable of modulating the functions of the protein complex or theVAP-A-interacting protein, e.g., by triggering or initiating, enhancingor stabilizing protein-protein interaction between the interactingprotein members of the protein complex, or the mutant forms of suchinteracting protein members found in the patient.

7. Cell and Animal Models

In another aspect of the present invention, cell and animal models areprovided in which one or more of the VAP-A-containing protein complexesidentified in the present invention, or VAP-A itself, or a member, ormembers of the group consisting of PPP1R3 and GTR3, exhibit aberrantfunction, activity, or concentration when compared with wildtype cellsand animals (e.g., increased or decreased concentration, alteredinteractions between protein complex constituents due to mutations ininteraction domains, and/or altered distribution or localization of theprotein complexes or constituents thereof in organs, tissues, cells, orcellular compartments). Such cell and animal models are useful tools forstudying cellular functions and biological processes associated with theprotein complexes of the present invention, or with VAP-A itself, orwith a VAP-A-interacting protein identified in accordance with thepresent invention. Such cell and animal models are also useful tools forstudying disorders and diseases associated with the protein complexes ofthe present invention, or VAP-A itself, or a member, or members of thegroup PPP1R3 and GTR3, and for testing various methods for modulatingthe cellular functions, and for treating the diseases and disorders,associated with aberrations in these protein complexes or the proteinconstituents thereof.

7.1. Cell Models

Cell models having an aberrant form of one or more of the proteincomplexes of the present invention are provided in accordance with thepresent invention.

The cell models may be established by isolating, from a patient, cellshaving an aberrant form of one or more of the protein complexes of thepresent invention. The isolated cells may be cultured in vitro as aprimary cell culture. Alternatively, the cells obtained from the primarycell culture or directly from the patient may be immortalized toestablish a human cell line. Any methods for constructing immortalizedhuman cell lines may be used in this respect. See generally Yeager andReddel, Curr. Opini. Biotech., 10:465-469 (1999). For example, the humancells may be immortalized by transfection of plasmids expressing theSV40 early region genes (See e.g., Jha et al., Exp. Cell Res., 245:1-7(1998)), introduction of the HPV E6 and E7 oncogenes (See e.g.,Reznikoff et al., Genes Dev., 8:2227-2240 (1994)), and infection withEpstein-Barr virus (See e.g., Tahara et al., Oncogene, 15:1911-1920(1997)). Alternatively, the human cells may be immortalized byrccombinantly expressing the gene for the human telomerase catalyticsubunit hTERT in the human cells. See Bodnar et al., Science,279:349-352 (1998).

In alternative embodiments, cell models are provided by recombinantlymanipulating appropriate host cells. The host cells may be bacteriacells, yeast cells, insect cells, plant cells, animal cells, and thelike. Preferably, the cells are derived from mammals, most preferablyhumans. The host cells may be obtained directly from an individual, or aprimary cell culture, or preferably an immortal stable human cell line.In a preferred embodiment, human embryonic stem cells or pluripotentcell lines derived from human stem cells are used as host cells. Methodsfor obtaining such cells are disclosed in, e.g., Shamblott, et al.,Proc. Natl. Acad. Sci. USA, 95:13726-13731 (1998) and Thomson et al.,Science, 282:1145-1147 (1998).

In one embodiment, a cell model is provided by recombinantly expressingone or more of the protein complexes of the present invention in cellsthat do not normally express such protein complexes. For example, cellsthat do not contain a particular protein complex may be engineered toexpress the protein complex. In a specific embodiment, a particularhuman protein complex is expressed in non-human cells. The cell modelmay be prepared by introducing into host cells nucleic acids encodingall interacting protein members required for the formation of aparticular protein complex, and expressing the protein members in thehost cells. For this purpose, the recombinant expression methodsdescribed in Section 2.2 may be used. In addition, the methods forintroducing nucleic acids into host cells disclosed in the context ofgene therapy in Section 6.2.2 may also be used.

In another embodiment, a cell model over-expressing one or more of theprotein complexes of the present invention is provided. The cell modelmay be established by increasing the expression level of one or more ofthe interacting protein members of the protein complexes. In a specificembodiment, all interacting protein members of a particular proteincomplex are over-expressed. The over-expression may be achieved byintroducing into host cells exogenous nucleic acids encoding theproteins to be over-expressed, and selecting those cells thatover-express the proteins. The expression of the exogenous nucleic acidsmay be transient or, preferably stable. The recombinant expressionmethods described in Section 2.2, and the methods for introducingnucleic acids into host cells disclosed in the context of gene therapyin Section 6.2.2 may be used. Alternatively, the gene activation methoddisclosed in U.S. Pat. No. 5,641,670 can be used. Any host cells may beemployed for establishing the cell model. Preferably, human cellslacking a protein complex to be over-expressed, or having a normalconcentration of the protein complex, are used as host cells. The hostcells may be obtained directly from an individual, or a primary cellculture, or preferably a stable immortal human cell line. In a preferredembodiment, human embryonic stem cells or pluripotent cell lines derivedfrom human stem cells are used as host cells. Methods for obtaining suchcells are disclosed in, e.g., Shamblott, et al., Proc. Natl. Acad. Sci.USA, 95:13726-13731 (1998), and Thomson et al., Science, 282:1145-1147(1998).

In yet another embodiment, a cell model expressing an abnormally lowlevel of one or more of the protein complexes of the present inventionis provided. Typically, the cell model is established by geneticallymanipulating cells that express a normal and detectable level of aprotein complex identified in accordance with the present invention.Generally the expression level of one or more of the interacting proteinmembers of the protein complex is reduced by recombinant methods. In aspecific embodiment, the expression of all interacting protein membersof a particular protein complex is reduced. The reduced expression maybe achieved by “knocking out” the genes encoding one or more interactingprotein members. Alternatively, mutations that can cause reducedexpression level (e.g., reduced transcription and/or translationefficiency, and decreased mRNA stability) may also be introduced intothe gene by homologous recombination. A gene encoding a ribozyme orantisense compound specific to the mRNA encoding an interacting proteinmember may also be introduced into the host cells, preferably stablyintegrated into the genome of the host cells. In addition, a geneencoding an antibody or fragment thereof specific to an interactingprotein member may also be introduced into the host cells. Therecombinant expression methods described in Sections 2.2, 6.1 and 6.2can all be used for purposes of manipulating the host cells.

The present invention also contemplates a cell model provided byrecombinant DNA techniques that exhibits aberrant interactions betweenthe interacting protein members of a protein complex identified in thepresent invention. For example, variants of the interacting proteinmembers of a particular protein complex exhibiting alteredprotein-protein interaction properties and the nucleic acid variantsencoding such variant proteins may be obtained by random orsite-directed mutagenesis in combination with a protein-proteininteraction assay system, particularly the yeast two-hybrid systemdescribed in Section 5.3. 1. Essentially, the genes encoding one or moreinteracting protein members of a particular protein complex may besubject to random or site-specific mutagenesis and the mutated genesequences are used in yeast two-hybrid system to test theprotein-protein interaction characteristics of the protein variantsencoded by the gene variants. In this manner, variants of theinteracting protein members of the protein complex may be identifiedthat exhibit altered protein-protein interaction properties in formingthe protein complex, e.g., increased or decreased binding affinity, andthe like. The nucleic acid variants encoding such protein variants maybe introduced into host cells by the methods described above, preferablyinto host cells that normally do not express the interacting proteins.

7.2. Cell-Based Assays

The cell models of the present invention containing an aberrant form ofa VAP-A-containing protein complex of the present invention are usefulin screening assays for identifying compounds useful in treatingdiseases and disorders involving docking and fusion of membrane vesiclesto target organelle membranes and cellular uptake of glucose such asdiabetes, obesity, ischemia, and insulin resistance. In addition, theymay also be used in in vitro pre-clinical assays for testing compounds,such as those identified in the screening assays of the presentinvention.

For example, cells may be treated with compounds to be tested andassayed for the compound's activity. A variety of parameters relevant toparticularly physiological disorders or diseases may be analyzed.

7.3. Transgenic Animals

In another aspect of the present invention, transgenic non-human animalsare created expressing an aberrant form of one or more of theVAP-A-containing protein complexes of the present invention. Animals ofany species may be used to generate the transgenic animal models,including but not limited to, mice, rats, hamsters, sheep, pigs,rabbits, guinea pigs, preferably non-human primates such as monkeys,chimpanzees, baboons, and the like.

In one embodiment, transgenic animals are made to over-express one ormore protein complexes formed from VAP-A, or a derivative, fragment orhomologue thereof (including the animal counterpart of VAP-A, i.e., anorthologue) and a member, or members, of the group of VAP-A-interactingproteins including PPP1R3 and GTR3, or derivatives, fragments orhomologues thereof (including orthologues). Over-expression may bedirected in a tissue or cell type that normally expresses animalcounterparts of such protein complexes. Consequently, the concentrationof the protein complex(es) will be elevated to higher levels thannormal. Alternatively, the one or more protein complexes are expressedin tissues or cells that do not normally express such proteins and hencedo not normally contain the protein complexes of the present invention.In a specific embodiment, human VAP-A and a human protein, or proteins,from the group of VAP-A-interacting proteins including PPP1R3 and GTR3,are expressed in the transgenic animals.

To achieve over-expression in transgenic animals, the transgenic animalsare made such that they contain and express exogenous, orthologous genesencoding VAP-A or a homologue, derivative or mutant form thereof and oneor more VAP-A-interacting proteins or homologues, derivatives or mutantforms thereof. Preferably, the exogenous genes are human genes. Suchexogenous genes may be operably linked to a native or non-nativepromoter, preferably a non-native promoter. For example, an exogenousVAP-A gene may be operably linked to a promoter that is not the nativeVAP-A promoter. If the expression of the exogenous gene is desired to belimited to a particular tissue, an appropriate tissue-specific promotermay be used.

Over-expression may also be achieved by manipulating the native promoterto create mutations that lead to gene over-expression, or by a geneactivation method such as that disclosed in U.S. Pat. No. 5,641,670 asdescribed above.

In another embodiment, the transgenic animal expresses an abnormally lowconcentration of the complex comprising VAP-A and one or more of theVAP-A-interacting proteins from the group PPP1R3 and GTR3. In a specificembodiment, the transgenic animal is a “knockout” animal wherein theendogenous gene encoding the animal orthologue of VAP-A and/or anendogenous gene encoding an animal orthologue of a VAP-A-interactingprotein are knocked out. In a specific embodiment, the expression of theanimal orthologues of both VAP-A and a VAP-A-interacting protein, orproteins, from the group PPP1R3 and GTR3 are reduced or knocked out. Thereduced expression may be achieved by knocking out the genes encodingone or both interacting protein members, typically by homologousrecombination. Alternatively, mutations that can cause reducedexpression (e.g., reduced transcription and/or translation efficiency,or decreased mRNA stability) may also be introduced into the endogenousgenes by homologous recombination. Genes encoding ribozymes or antisensecompounds specific to the mRNAs encoding the interacting protein membersmay also be introduced into the transgenic animal. In addition, genesencoding antibodies or fragments thereof specific to the interactingprotein members may also be introduced into the transgenic animal.

In an alternate embodiment, transgenic animals are made in which theendogenous genes encoding the animal orthologues of VAP-A and one ormore VAP-A-interacting proteins from the group PPP1R3 and GTR3 arereplaced with orthologous human genes.

In yet another embodiment, the transgenic animal of this inventionexpresses specific mutant forms of VAP-A and one or moreVAP-A-interacting proteins from the group PPP1R3 and GTR3 that exhibitaberrant interactions. For this purpose, variants of VAP-A and one ormore VAP-A-interacting proteins from the group PPP1R3 and GTR3exhibiting altered protein-protein interaction properties, and thenucleic acid variants encoding such variant proteins, may be obtained byrandom or site-specific mutagenesis in combination with aprotein-protein interaction assay system, particularly the yeasttwo-hybrid system described in Section 5.3. 1. For example, variants ofVAP-A and PPP1R3 exhibiting increased, decreased or abolished bindingaffinity to each other may be identified and isolated. The transgenicanimal of the present invention may be made to express such proteinvariants by modifying the endogenous genes. Alternatively, the nucleicacid variants may be introduced exogenously into the transgenic animalgenome to express the protein variants therein. In a specificembodiment, the exogenous nucleic acid variants are derived fromorthologous human genes and the corresponding endogenous genes areknocked out.

Any techniques known in the art for making transgenic animals may beused for purposes of the present invention. For example, the transgenicanimals of the present invention may be provided by methods describedin, e.g., Jaenisch, Science, 240:1468-1474 (1988); Capecchi, et al.,Science, 244:1288-1291 (1989); Hasty et al., Nature, 350:243 (1991);Shinkai et al., Cell, 68:855 (1992); Mombaerts et al., Cell, 68:869(1992); Philpott et al., Science, 256:1448 (1992); Snouwaert et al.,Science, 257:1083 (1992); Donehower et al., Nature, 356:215 (1992);Hogan et al., Manipulating the Mouse Embryo; A Laboratory Manual, 2^(nd)edition, Cold Spring Harbor Laboratory Press, 1994; and U.S. Pat. Nos.4,873,191; 5,800,998; 5,891,628, all of which are incorporated herein byreference.

Generally, the founder lines may be established by introducingappropriate exogenous nucleic acids into, or modifying an endogenousgene in, germ lines, embryonic stem cells, embryos, or sperm which arethen used in producing a transgenic animal. The gene introduction may beconducted by various methods including those described in Sections 2.2,6.1 and 6.2. See also, Van der Putten et al., Proc. Natl. Acad. Sci.USA, 82:6148-6152 (1985); Thompson et al., Cell, 56:313-321 (1989); Lo,Mol. Cell. Biol., 3:1803-1814 (1983); Gordon, Trangenic Animals, Intl.Rev. Cytol. 115:171-229 (1989); and Lavitrano et al., Cell, 57:717-723(1989). In a specific embodiment, the exogenous gene is incorporatedinto an appropriate vector, such as those described in Sections 2.2 and6.2, and is transformed into embryonic stem (ES) cells. The transformedES cells are then injected into a blastocyst. The blastocyst with thetransformed ES cells is then implanted into a surrogate mother animal.In this manner, a chimeric founder line animal containing the exogenousnucleic acid (transgene) may be produced.

Preferably, site-specific recombination is employed to integrate theexogenous gene into a specific predetermined site in the animal genome,or to replace an endogenous gene or a portion thereof with the exogenoussequence. Various site-specific recombination systems may be usedincluding those disclosed in Sauer, Curr. Opin. Biotechnol., 5:521-527(1994); Capecchi, et al., Science, 244:1288-1291 (1989); and Gu et al.,Science, 265:103-106 (1994). Specifically, the Cre/lox site-specificrecombination system known in the art may be conveniently used whichemploys the bacteriophage P1 protein Cre recombinase and its recognitionsequence loxP. See Rajewsky et al., J. Clin. Invest., 98:600-603 (1996);Sauer, Methods, 14:381-392 (1998); Gu et al., Cell, 73:1155-1164 (1993);Araki et al., Proc. Natl. Acad. Sci. USA, 92:160-164 (1995); Lakso etal., Proc. Natl. Acad. Sci. USA, 89:6232-6236 (1992); and Orban et al.,Proc. Natl. Acad. Sci. USA, 89:6861-6865 (1992).

The transgenic animals of the present invention may be transgenicanimals that carry a transgene in all cells or mosaic transgenic animalscarrying a transgene only in certain cells, e.g., somatic cells. Thetransgenic animals may have a single copy or multiple copies of aparticular transgene.

The founder transgenic animals thus produced may be bred to producevarious offsprings. For example, they can be inbred, outbred, andcrossbred to establish homozygous lines, heterozygous lines, andcompound homozygous or heterozygous lines.

8. Pharmaceutical Compositions and Formulations

In another aspect of the present invention, pharmaceutical compositionsare also provided containing one or more of the therapeutic agentsprovided in the present invention as described in Section 6. Thecompositions are prepared as a pharmaceutical formulation suitable foradministration into a patient. Accordingly, the present invention alsoextends to pharmaceutical compositions, medicaments, drugs or othercompositions containing one or more of the therapeutic agent inaccordance with the present invention.

For example, such therapeutic agents include, but are not limited to,(1) small organic compounds selected based on the screening methods ofthe present invention capable of interfering with the interactionbetween VAP-A and an interactor thereof, (2) antisense compoundsspecifically hybridizable to VAP-A nucleic acids (gene or mRNA) (3)antisense compounds specific to the gene or mRNA of a VAP-A-interactingprotein, (4) ribozyme compounds specific to VAP-A nucleic acids (gene ormRNA), (5) ribozyme compounds specific to the gene or mRNA of aVAP-A-interacting protein, (6) antibodies immunoreactive with VAP-A or aVAP-A-interacting protein, (7) antibodies selectively immunoreactivewith a protein complex of the present invention, (8) small organiccompounds capable of binding a protein complex of the present invention,(9) small peptide compounds as described above (optionally linked to atransporter) capable of interacting with VAP-A or a VAP-A-interactingprotein, (10) nucleic acids encoding the antibodies or peptides, etc.

The compositions are prepared as a pharmaceutical formulation suitablefor administration into a patient. Accordingly, the present inventionalso extends to pharmaceutical compositions, medicaments, drugs or othercompositions containing one or more of the therapeutic agent inaccordance with the present invention.

In the pharmaceutical composition, an active compound identified inaccordance with the present invention can be in any pharmaceuticallyacceptable salt form. As used herein, the term “pharmaceuticallyacceptable salts” refers to the relatively non-toxic, organic orinorganic salts of the compounds of the present invention, includinginorganic or organic acid addition salts of the compound. Examples ofsuch salts include, but are not limited to, hydrochloride salts, sulfatesalts, bisulfate salts, borate salts, nitrate salts, acetate salts,phosphate salts, hydrobromide salts, laurylsulfonate salts,glucoheptonate salts, oxalate salts, oleate salts, laurate salts,stearate salts, palmitate salts, valerate salts, benzoate salts,naththylate salts, mesylate salts, tosylate salts, citrate salts,lactate salts, maleate salts, succinate salts, tartrate salts, fumaratesalts, and the like. See, e.g., Berge, et al., J. Pharm. Sci., 66:1-19(1977).

For oral delivery, the active compounds can be incorporated into aformulation that includes pharmaceutically acceptable carriers such asbinders (e.g., gelatin, cellulose, gum tragacanth), excipients (e.g.,starch, lactose), lubricants (e.g., magnesium stearate, silicondioxide), disintegrating agents (e.g., alginate, Primogel, and cornstarch), and sweetening or flavoring agents (e.g., glucose, sucrose,saccharin, methyl salicylate, and peppermint). The formulation can beorally delivered in the form of enclosed gelatin capsules or compressedtablets. Capsules and tablets can be prepared in any conventionaltechniques. The capsules and tablets can also be coated with variouscoatings known in the art to modify the flavors, tastes, colors, andshapes of the capsules and tablets. In addition, liquid carriers such asfatty oil can also be included in capsules.

Suitable oral formulations can also be in the form of suspension, syrup,chewing gum, wafer, elixir, and the like. If desired, conventionalagents for modifying flavors, tastes, colors, and shapes of the specialforms can also be included. In addition, for convenient administrationby enteral feeding tube in patients unable to swallow, the activecompounds can be dissolved in an acceptable lipophilic vegetable oilvehicle such as olive oil, corn oil and safflower oil.

The active compounds can also be administered parenterally in the formof solution or suspension, or in lyophilized form capable of conversioninto a solution or suspension form before use. In such formulations,diluents or pharmaceutically acceptable carriers such as sterile waterand physiological saline buffer can be used. Other conventionalsolvents, pH buffers, stabilizers, anti-bacterial agents, surfactants,and antioxidants can all be included. For example, useful componentsinclude sodium chloride, acetate, citrate or phosphate buffers,glycerin, dextrose, fixed oils, methyl parabens, polyethylene glycol,propylene glycol, sodium bisulfate, benzyl alcohol, ascorbic acid, andthe like. The parenteral formulations can be stored in any conventionalcontainers such as vials and ampoules.

Routes of topical administration include nasal, bucal, mucosal, rectal,or vaginal applications. For topical administration, the activecompounds can be formulated into lotions, creams, ointments, gels,powders, pastes, sprays, suspensions, drops and aerosols. Thus, one ormore thickening agents, humectants, and stabilizing agents can beincluded in the formulations. Examples of such agents include, but arenot limited to, polyethylene glycol, sorbitol, xanthan gum, petrolatum,beeswax, or mineral oil, lanolin, squalene, and the like. A special formof topical administration is delivery by a transdermal patch. Methodsfor preparing transdermal patches are disclosed, e.g., in Brown, et al.,Annual Review of Medicine, 39:221-229 (1988), which is incorporatedherein by reference.

Subcutaneous implantation for sustained release of the active compoundsmay also be a suitable route of administration. This entails surgicalprocedures for implanting an active compound in any suitable formulationinto a subcutaneous space, e.g., beneath the anterior abdominal wall.See, e.g., Wilson et al., J. Clin. Psych. 45:242-247 (1984). Hydrogelscan be used as a carrier for the sustained release of the activecompounds. Hydrogels are generally known in the art. They are typicallymade by crosslinking high molecular weight biocompatible polymers into anetwork that swells in water to form a gel like material. Preferably,hydrogels is biodegradable or biosorbable. For purposes of thisinvention, hydrogels made of polyethylene glycols, collagen, orpoly(glycolic-co-L-lactic acid) may be useful. See, e.g., Phillips etal., J. Pharmaceut. Sci. 73:1718-1720 (1984).

The active compounds can also be conjugated, to a water solublenon-immunogenic non-peptide high molecular weight polymer to form apolymer conjugate. For example, an active compound is covalently linkedto polyethylene glycol to form a conjugate. Typically, such a conjugateexhibits improved solubility, stability, and reduced toxicity andimmunogenicity. Thus, when administered to a patient, the activecompound in the conjugate can have a longer half-life in the body, andexhibit better efficacy. See generally, Burnham, Am. J. Hosp. Pharm.,15:210-218 (1994). PEGylated proteins are currently being used inprotein replacement therapies and for other therapeutic uses. Forexample, PEGylated interferon (PEG-INTRON A®) is clinically used fortreating Hepatitis B. PEGylated adenosine deaminase (ADAGEN®) is beingused to treat severe combined immunodeficiency disease (SCIDS).PEGylated L-asparaginase (ONCAPSPAR®) is being used to treat acutelymphoblastic leukemia (ALL). It is preferred that the covalent linkagebetween the polymer and the active compound and/or the polymer itself ishydrolytically degradable under physiological conditions. Suchconjugates known as “prodrugs” can readily release the active compoundinside the body. Controlled release of an active compound can also beachieved by incorporating the active ingredient into microcapsules,nanocapsules, or hydrogels generally known in the art.

Liposomes can also be used as carriers for the active compounds of thepresent invention. Liposomes are micelles made of various lipids such ascholesterol, phospholipids, fatty acids, and derivatives thereof.Various modified lipids can also be used. Liposomes can reduce thetoxicity of the active compounds, and increase their stability. Methodsfor preparing liposomal suspensions containing active ingredientstherein are generally known in the art. See, e.g., U.S. Pat. No.4,522,811; Prescott, Ed., Methods in Cell Biology, Volume XIV, AcademicPress, New York, N.Y. (1976).

The active compounds can also be administered in combination withanother active agent that synergistically treats or prevents the samesymptoms or is effective for another disease or symptom in the patienttreated so long as the other active agent does not interfere with oradversely affect the effects of the active compounds of this invention.Such other active agents include but are not limited toanti-inflammation agents, antiviral agents, antibiotics, antifungalagents, antithrombotic agents, cardiovascular drugs, cholesterollowering agents, anti-cancer drugs, hypertension drugs, and the like.

Generally, the toxicity profile and therapeutic efficacy of thetherapeutic agents can be determined by standard pharmaceuticalprocedures in cell models or animal models, e.g., those provided inSection 7. As is known in the art, the LD₅₀ represents the dose lethalto about 50% of a tested population. The ED₅₀ is a parameter indicatingthe dose therapeutically effective in about 50% of a tested population.Both LD₅₀ and ED₅₀ can be determined in cell models and animal models.In addition, the IC₅₀ may also be obtained in cell models and animalmodels, which stands for the circulating plasma concentration that iseffective in achieving about 50% of the maximal inhibition of thesymptoms of a disease or disorder. Such data may be used in designing adosage range for clinical trials in humans. Typically, as will beapparent to skilled artisans, the dosage range for human use should bedesigned such that the range centers around the ED₅₀ and/or IC₅₀, butsignificantly below the LD₅₀ obtained from cell or animal models.

It will be apparent to skilled artisans that therapeutically effectiveamount for each active compound to be included in a pharmaceuticalcomposition of the present invention can vary with factors including butnot limited to the activity of the compound used, stability of theactive compound in the patient's body, the severity of the conditions tobe alleviated, the total weight of the patient treated, the route ofadministration, the ease of absorption, distribution, and excretion ofthe active compound by the body, the age and sensitivity of the patientto be treated, and the like. The amount of administration can also beadjusted as the various factors change over time.

EXAMPLES

1. Yeast Two-Hybrid System

The principles and methods of the yeast two-hybrid system have beendescribed in detail in The Yeast Two-Hybrid System, Bartel and Fields,eds., pages 183-196, Oxford University Press, New York, N.Y., 1997. Thefollowing is thus a description of the particular procedure that weused, which was applied to all proteins.

The cDNA encoding the bait protein was generated by PCR from cDNAprepared from a desired tissue. The cDNA product was then introduced byrecombination into the yeast expression vector pGBT.Q, which is a closederivative of pGBT.C (See Bartel et al., Nat Genet., 12:72-77 (1996)) inwhich the polylinker site has been modified to include M13 sequencingsites. The new construct was selected directly in the yeast strainPNY200 for its ability to drive tryptophane synthesis (genotype of thisstrain: MAT α trp1-901 leu2-3,112 ura3-52 his3-200 ade2 gal4Δ gal80). Inthese yeast cells, the bait was produced as a C-terminal fusion proteinwith the DNA binding domain of the transcription factor Gal4 (aminoacids 1 to 147). Prey libraries were transformed into the yeast strainBK100 (genotype of this strain: MATa trp1-901 leu2-3,112 ura3-52his3-200 gal4Δ gal80 LYS2::GAL-HIS3 GAL2-ADE2 met2::GAL7-lacZ), andselected for the ability to drive leucine synthesis. In these yeastcells, each cDNA was expressed as a fusion protein with thetranscription activation domain of the transcription factor Gal4 (aminoacids 768 to 881) and a 9 amino acid hemagglutinin epitope tag. PNY200cells (MATα mating type), expressing the bait, were then mated withBK100 cells (MATa mating type), expressing prey proteins from a preylibrary. The resulting diploid yeast cells expressing proteinsinteracting with the bait protein were selected for the ability tosynthesize tryptophan, leucine, histidine, and adenine. DNA was preparedfrom each clone, transformed by electroporation into E. coli strain KC8(Clontech KC8 electrocompetent cells, Catalog No. C2023-1), and thecells were selected on ampicillin-containing plates in the absence ofeither tryptophane (selection for the bait plasmid) or leucine(selection for the library plasmid). DNA for both plasmids was preparedand sequenced by the dideoxynucleotide chain termination method. Theidentity of the bait cDNA insert was confirmed and the cDNA insert fromthe prey library plasmid was identified using the BLAST program tosearch against public nucleotide and protein databases. Plasmids fromthe prey library were then individually transformed into yeast cellstogether with a plasmid driving the synthesis of lamin and 5 other testproteins, respectively, fused to the Gal4 DNA binding domain. Clonesthat gave a positive signal in the β-galactosidase assay were consideredfalse-positives and discarded. Plasmids for the remaining clones weretransformed into yeast cells together with the original bait plasmid.Clones that gave a positive signal in the β-galactosidase assay wereconsidered true positives.

Bait sequences indicated in Table I were used in the yeast two-hybridsystem described above. The isolated prey sequences are summarized inTable I. The GenBank Accession Nos. for the bait and prey proteins arealso provided in Table I, upon which the bait and prey sequences arealigned.

2. Production of Antibodies Selectively Immunoreactive with ProteinComplex

The VAP-A-interacting region of PPP1R3 and the PPP1R3-interacting regionof VAP-A are indicated in Table I in Section 2. Both regions, orfragments thereof, are recombinantly-expressed in E. coli. and isolatedand purified. Mixing the two purified interacting regions forms aprotein complex. A protein complex is also formed by mixingrecombinantly expressed intact complete VAP-A and PPP1R3. The twoprotein complexes are used as antigens in immunizing a mouse. mRNA isisolated from the immunized mouse spleen cells, and first-strand cDNA issynthesized using the mRNA as a template. The V_(H) and V_(K) genes areamplified from the thus synthesized cDNAs by PCR using appropriateprimers.

The amplified V_(H) and V_(K) genes are ligated together and subclonedinto a phagemid vector for the construction of a phage display library.E. coli. cells are transformed with the ligation mixtures, and thus aphage display library is established. Alternatively, the ligated V_(H)and V_(k) genes are subcloned into a vector suitable for ribosomedisplay in which the V_(H)-V_(k) sequence is under the control of a T7promoter. See Schaffitzel et al., J. Immun. Meth., 231:119-135 (1999).

The libraries are screened for their ability to bind VAP-A-PPP1R3complex and VAP-A or PPP1R3, alone. Several rounds of screening aregenerally performed. Clones corresponding to scFv fragments that bindthe VAP-A-PPP1R3 complex, but not isolated VAP-A or PPP1R3 are selectedand purified. A single purified clonc is used to prepare an antibodyselectively immunoreactive with the complex comprising VAP-A and PPP1R3.The antibody is then verified by an immunochemistry method such as RIAand ELISA.

In addition, the clones corresponding to scFv fragments that bind thecomplex comprising VAP-A and PPP1R3, and also bind isolated VAP-A and/orPPP1R3 may be selected. The scFv genes in the clones are diversified bymutagenesis methods such as oligonucleotide-directed mutagenesis,error-prone PCR (See Lin-Goerke et al., Biotechniques, 23:409 (1997)),dNTP analogues (See Zaccolo et al., J. Mol. Biol., 255:589 (1996)), andother methods. The diversified clones are further screened in phagedisplay or ribosome display libraries. In this manner, scFv fragmentsselectively immunoreactive with the complex comprising VAP-A and PPP1R3may be obtained.

3. Yeast Screen to Identify Small Molecule Inhibitors of the InteractionBetween VAP-A and PPP1R3

Beta-galactosidase is used as a reporter enzyme to signal theinteraction between yeast two-hybrid protein pairs expressed fromplasmids in Saccharomyces cerevisiae. Yeast strain MY209 (ade2 his3 leu2trp1cyh2 ura3::GAL1p-lacZ gal4 gal80 lys2::GAL1p-HIS3) bearing oneplasmid with the genotype of LEU2 CEN4 ARS1 ADH1p-SV40NLS-GAL4 (768-881)-PPP1R3-PGK1t AmpR ColE1_ori, and another plasmid having a genotype ofTRP1 CEN4 ARS ADH1 p-GAL4(1-147)-VAP-A-ADH1t AmpR ColE1_ori is culturedin synthetic complete media lacking leucine and tryptophan (SC-Leu-Trp)overnight at 30° C. The VAP-A and PPP1R3 nucleic acids in the plasmidscan code for the full-length VAP-A and PPP1R3 proteins, respectively, orfragments thereof. This culture is diluted to 0.01 OD₆₃₀ units/ml usingSC-Leu-Trp media. The diluted MY209 culture is dispensed into 96-wellmicroplates. Compounds from a library of small molecules are added tothe microplates; the final concentration of test compounds isapproximately 60 μM. The assay plates are incubated at 30° C. overnight.

The following day an aliquot of concentrated substrate/lysis buffer isadded to each well and the plates incubated at 37° C. for 1-2 hours. Atan appropriate time an aliquot of stop solution is added to each well tohalt the beta-galactosidase reaction. For all microplates an absorbancereading is obtained to assay the generation of product from the enzymesubstrate. The presence of putative inhibitors of the interactionbetween VAP-A and PPP1R3 results in inhibition of the beta-galactosidasesignal generated by MY209. Additional testing eliminates compounds thatdecreased expression of beta-galactosidase by affecting yeast cellgrowth and non-specific inhibitors that affected the beta-galactosidasesignal generated by the interaction of an unrelated protein pair.

Once a hit, i.e., a compound which inhibits the interaction between theinteracting proteins, is obtained, the compound is identified andsubjected to further testing wherein the compounds are assayed atseveral concentrations to determine an IC₅₀ value, this being theconcentration of the compound at which the signal seen in the two-hybridassay described in this Example is 50% of the signal seen in the absenceof the inhibitor.

4. Enzyme-Linked Immunosorbent Assay (ELISA)

pGEX5X-2 (Amersham Biosciences; Uppsala, Sweden) is used for theexpression of a GST-PPP1R3 fusion protein. The pGEX5X-2-PPP1R3 constructis transfected into Escherichia coli strain DH5α (Invitrogen; Carlsbad,Calif.) and fusion protein is prepared by inducing log phase cells (O.D.595=0.4) with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG).Cultures are harvested after approximately 4 hours of induction, andcells pelleted by centrifugation. Cell pellets are resuspended in lysisbuffer (1% nonidet P-40 [NP-40], 150 mM NaCl, 10 mM Tris pH 7.4, 1 mMABESF [4-(2-aminoethyl)benzenesulfonyl fluoride]), lysed by sonicationand the lysate cleared of insoluble materials by centrifugation. Clearedlysate is incubated with Glutathione Sepharose beads (AmershamBiosciences; Uppsala, Sweden) followed by thorough washing with lysisbuffer. The GST-PPP1R3 fusion protein is then eluted from the beads with5 mM reduced glutathione. Eluted protein is dialyzed against phosphatebuffer saline (PBS) to remove the reduced glutathione.

A stable Drosophila Schneider 2 (S2) myc-VAP-A expression cell line isgenerated by transfecting S2 cells with the hygromycin B resistancevector PCOHYGRO™ (Invitrogen; Carlsbad, Calif.) and an expression vectorthat directs the expression of the myc-VAP-A fusion protein. Briefly, S2cells are washed and re-suspended in serum free Express Five media(Invitrogen; Carlsbad, Calif.). Plasmid/liposome complexes are thenadded (NOVAFECTOR™; Venn Nova; Pompano Beach, Fla.) and allowed toincubate with cells for 12 hours under standard growth conditions (roomtemperature, no CO₂buffering). Following this incubation period fetalbovine serum is added to a final concentration of 20% and cells areallowed to recover for 24 hours. The media is replaced and cells aregrown for an additional 24 hours. Transfected cells are then selected in350 μg/ml hygromycin for three weeks. Expression of myc-VAP-A isconfirmed by Western blotting. This cell line is referred to asS2-myc-VAP-A.

GST-PPP1R3 fusion protein is immobilized to wells of an ELISA plate asfollows: high absorption microtiter (NUNC MAXISORB™ 96 well) ELISAplates (Nalge Nunc International; Rochester, N.Y.) are incubated with100 μl of 10 μg/ml of GST-PPP1R3 in 50 mM cabonate buffer (pH 9.6) andstored overnight at 4° Celsius. This plate is referred to as the ELISAplate.

A compound dilution plate is generated in the following manner. In a 96well polypropylene plate (Greiner, Germany) 50 μl of DMSO is pipettedinto columns 2-12. In the same polypropylene plate pipette, 10 μl ofeach compound being tested for its ability to modulate protein-proteininteractions is plated in the wells of column 1 followed by 90 μl ofDMSO (final volume of 100 μl). Compounds selected from primary screensor from virtual screening, or designed based on the primary screen hitsare then serially diluted by removing 50 μl from column 1 andtransferring it to column 2 (50:50 dilution). Serial dilutions arecontinued until column 10. This plate is termed the compound dilutionplate.

Next, 12 μl from each well of the compound dilution plate is transferredinto its corresponding well in a new polypropylene plate. 108 μl ofS2-myc-VAP-A-containing lysate (1×10⁶ cell equivalents/ml) in phosphatebuffered saline is added to all wells of columns 1-11 . 108 μl ofphosphate buffered saline without lysate is added into all wells ofcolumn 12. The plate is then mixed on a shaker for 15 minutes. Thisplate is referred to as the compound preincubation plate.

The ELISA plate is emptied of its contents and 400 μl of Superblock(Pierce Endogen; Rockford, Ill.) is added to all the wells and allowedto sit for 1 hour at room temperature. 100 μl from all columns of thecompound preincubation plate are transferred into the correspondingwells of the ELISA binding plate. The plate is then covered and allowedto incubate for 1.5 hours room temperature.

The interaction of the myc-tagged VAP-A with the immobilized GST-PPP1R3is detected by washing the ELISA plate followed by an incubation with100 μl/well of 1 μg/ml of mouse anti-myc IgG (clone 9E10; Roche AppliedScience; Indianapolis, Ind.) in phosphate buffered saline. After 1 hourat room temperature, the plates are washed with phosphate bufferedsaline and incubated with 100 μl/well of 250 ng/ml of goat anti-mouseIgG conjugated to horseradish peroxidase in phosphate buffer saline.Plates are then washed again with phosphate buffered saline andincubated with the fluorescent substrate solution Quantiblu (PierceEndogen; Rockford, Ill.). Horseradish peroxidase activity is thenmeasured by reading the plates in a fluorescent plate reader (325 nmexcitation, 420 nm emission).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

In various parts of this disclosure, certain publications or patents arediscussed or cited. The mere discussion of, or reference to, suchpublications or patents is not intended as admission that they are priorart to the present invention.

3 1 242 PRT Homo sapiens 1 Met Ala Asn Asp Glu Gln Ile Leu Val Leu AspPro Pro Thr Asp Leu 1 5 10 15 Lys Phe Lys Gly Pro Phe Thr Asp Val ValThr Thr Asn Leu Lys Leu 20 25 30 Arg Asn Pro Ser Asp Arg Lys Val Cys PheLys Val Lys Thr Thr Ala 35 40 45 Pro Arg Arg Tyr Cys Val Arg Pro Asn SerGly Ile Ile Asp Pro Gly 50 55 60 Ser Thr Val Thr Val Ser Val Met Leu GlnPro Phe Asp Tyr Asp Pro 65 70 75 80 Asn Glu Lys Ser Lys His Lys Phe MetVal Gln Thr Ile Phe Ala Pro 85 90 95 Pro Asn Thr Ser Asp Met Glu Ala ValTrp Lys Glu Ala Lys Pro Asp 100 105 110 Glu Leu Met Asp Ser Lys Leu ArgCys Val Phe Glu Met Pro Asn Glu 115 120 125 Asn Asp Lys Leu Asn Asp MetGlu Pro Ser Lys Ala Val Pro Leu Asn 130 135 140 Ala Ser Lys Gln Asp GlyPro Met Pro Lys Pro His Ser Val Ser Leu 145 150 155 160 Asn Asp Thr GluThr Arg Lys Leu Met Glu Glu Cys Lys Arg Leu Gln 165 170 175 Gly Glu MetMet Lys Leu Ser Glu Glu Asn Arg His Leu Arg Asp Glu 180 185 190 Gly LeuArg Leu Arg Lys Val Ala His Ser Asp Lys Pro Gly Ser Thr 195 200 205 SerThr Ala Ser Phe Arg Asp Asn Val Thr Ser Pro Leu Pro Ser Leu 210 215 220Leu Val Val Ile Ala Ala Ile Phe Ile Gly Phe Phe Leu Gly Lys Phe 225 230235 240 Ile Leu 2 1122 PRT Homo sapiens 2 Met Glu Pro Ser Glu Val ProSer Gln Ile Ser Lys Asp Asn Phe Leu 1 5 10 15 Glu Val Pro Asn Leu SerAsp Ser Leu Cys Glu Asp Glu Glu Val Thr 20 25 30 Phe Gln Pro Gly Phe SerPro Gln Pro Ser Arg Arg Gly Ser Asp Ser 35 40 45 Ser Glu Asp Ile Tyr LeuAsp Thr Pro Ser Ser Gly Thr Arg Arg Val 50 55 60 Ser Phe Ala Asp Ser PheGly Phe Asn Leu Val Ser Val Lys Glu Phe 65 70 75 80 Asp Cys Trp Glu LeuPro Ser Ala Ser Thr Thr Phe Asp Leu Gly Thr 85 90 95 Asp Ile Phe His ThrGlu Glu Tyr Val Leu Ala Pro Leu Phe Asp Leu 100 105 110 Pro Ser Ser LysGlu Asp Leu Met Gln Gln Leu Gln Ile Gln Lys Ala 115 120 125 Ile Leu GluSer Thr Glu Ser Leu Leu Gly Ser Thr Ser Ile Lys Gly 130 135 140 Ile IleArg Val Leu Asn Val Ser Phe Glu Lys Leu Val Tyr Val Arg 145 150 155 160Met Ser Leu Asp Asp Trp Gln Thr His Tyr Asp Ile Leu Ala Glu Tyr 165 170175 Val Pro Asn Ser Cys Asp Gly Glu Thr Asp Gln Phe Ser Phe Lys Ile 180185 190 Val Leu Val Pro Pro Tyr Gln Lys Asp Gly Ser Lys Val Glu Phe Cys195 200 205 Ile Arg Tyr Glu Thr Ser Val Gly Thr Phe Trp Ser Asn Asn AsnGly 210 215 220 Thr Asn Tyr Thr Phe Ile Cys Gln Lys Lys Glu Gln Glu ProGlu Pro 225 230 235 240 Val Lys Pro Trp Lys Glu Val Pro Asn Arg Gln IleLys Gly Cys Leu 245 250 255 Lys Val Lys Ser Ser Lys Glu Glu Ser Ser ValThr Ser Glu Glu Asn 260 265 270 Asn Phe Glu Asn Pro Lys Asn Thr Asp ThrTyr Ile Pro Thr Ile Ile 275 280 285 Cys Ser His Glu Asp Lys Glu Asp LeuGlu Ala Ser Asn Arg Asn Val 290 295 300 Lys Asp Val Asn Arg Glu His AspGlu His Asn Glu Lys Glu Leu Glu 305 310 315 320 Leu Met Ile Asn Gln HisLeu Ile Arg Thr Arg Ser Thr Ala Ser Arg 325 330 335 Asp Glu Arg Asn ThrPhe Ser Thr Asp Pro Val Asn Phe Pro Asn Lys 340 345 350 Ala Glu Gly LeuGlu Lys Lys Gln Ile His Gly Glu Ile Cys Thr Asp 355 360 365 Leu Phe GlnArg Ser Leu Ser Pro Ser Ser Ser Ala Glu Ser Ser Val 370 375 380 Lys GlyAsp Phe Tyr Cys Asn Glu Lys Tyr Ser Ser Gly Asp Asp Cys 385 390 395 400Thr His Gln Pro Ser Glu Glu Thr Thr Ser Asn Met Gly Glu Ile Lys 405 410415 Pro Ser Leu Gly Asp Thr Ser Ser Asp Glu Leu Val Gln Leu His Thr 420425 430 Gly Ser Lys Glu Val Leu Asp Asp Asn Ala Asn Pro Ala His Gly Asn435 440 445 Gly Thr Met Gln Ile Pro Cys Pro Ser Ser Asp Gln Leu Met AlaGly 450 455 460 Asn Leu Asn Lys Lys His Glu Gly Gly Ala Lys Lys Ile GluVal Lys 465 470 475 480 Asp Leu Gly Cys Leu Arg Arg Asp Phe His Ser AspThr Ser Ala Cys 485 490 495 Leu Lys Glu Ser Thr Glu Glu Gly Ser Ser LysGlu Asp Tyr Tyr Gly 500 505 510 Asn Gly Lys Asp Asp Glu Glu Gln Arg IleTyr Leu Gly Val Asn Glu 515 520 525 Lys Gln Arg Lys Asn Phe Gln Thr IleLeu His Asp Gln Glu Arg Lys 530 535 540 Met Gly Asn Pro Lys Ile Ser ValAla Gly Ile Gly Ala Ser Asn Arg 545 550 555 560 Asp Leu Ala Thr Leu LeuSer Glu His Thr Ala Ile Pro Thr Arg Ala 565 570 575 Ile Thr Ala Asp ValSer His Ser Pro Arg Thr Asn Leu Ser Trp Glu 580 585 590 Glu Ala Val LeuThr Pro Glu His His His Leu Thr Ser Glu Gly Ser 595 600 605 Ala Leu GlyGly Ile Thr Gly Gln Val Cys Ser Ser Arg Thr Gly Asn 610 615 620 Val LeuArg Asn Asp Tyr Leu Phe Gln Val Glu Glu Lys Ser Gly Gly 625 630 635 640Ile Asn Ser Glu Asp Gln Asp Asn Ser Pro Gln His Lys Gln Ser Trp 645 650655 Asn Val Leu Glu Ser Gln Gly Lys Ser Arg Glu Asn Lys Thr Asn Ile 660665 670 Thr Glu His Ile Lys Gly Gln Thr Asp Cys Glu Asp Val Trp Gly Lys675 680 685 Arg Asp Asn Thr Arg Ser Leu Lys Ala Thr Thr Glu Glu Leu PheThr 690 695 700 Cys Gln Glu Thr Val Cys Cys Glu Leu Ser Ser Leu Ala AspHis Gly 705 710 715 720 Ile Thr Glu Lys Ala Glu Ala Gly Thr Ala Tyr IleIle Lys Thr Thr 725 730 735 Ser Glu Ser Thr Pro Glu Ser Met Ser Ala ArgGlu Lys Ala Ile Ile 740 745 750 Ala Lys Leu Pro Gln Glu Thr Ala Arg SerAsp Arg Pro Ile Glu Val 755 760 765 Lys Glu Thr Ala Phe Asp Pro His GluGly Arg Asn Asp Asp Ser His 770 775 780 Tyr Thr Leu Cys Gln Arg Asp ThrVal Gly Val Ile Tyr Asp Asn Asp 785 790 795 800 Phe Glu Lys Glu Ser ArgLeu Gly Ile Cys Asn Val Arg Val Asp Glu 805 810 815 Met Glu Lys Glu GluThr Met Ser Met Tyr Asn Pro Arg Lys Thr His 820 825 830 Asp Arg Glu LysCys Gly Thr Gly Asn Ile Thr Ser Val Glu Glu Ser 835 840 845 Ser Trp ValIle Thr Glu Tyr Gln Lys Ala Thr Ser Lys Leu Asp Leu 850 855 860 Gln LeuGly Met Leu Pro Thr Asp Lys Thr Val Phe Ser Glu Asn Arg 865 870 875 880Asp His Arg Gln Val Gln Glu Leu Ser Lys Lys Thr Asp Ser Asp Ala 885 890895 Ile Val His Ser Ala Phe Asn Ser Asp Thr Asn Arg Ala Pro Gln Asn 900905 910 Ser Ser Pro Phe Ser Lys His His Thr Glu Ile Ser Val Ser Thr Asn915 920 925 Glu Gln Ala Ile Ala Val Glu Asn Ala Val Thr Thr Met Ala SerGln 930 935 940 Pro Ile Ser Thr Lys Ser Glu Asn Ile Cys Asn Ser Thr ArgGlu Ile 945 950 955 960 Gln Gly Ile Glu Lys His Pro Tyr Pro Glu Ser LysPro Glu Glu Val 965 970 975 Ser Arg Ser Ser Gly Ile Val Thr Ser Gly SerArg Lys Glu Arg Cys 980 985 990 Ile Gly Gln Ile Phe Gln Thr Glu Glu TyrSer Val Glu Lys Ser Leu 995 1000 1005 Gly Pro Met Ile Leu Ile Asn LysPro Leu Glu Asn Met Glu Glu 1010 1015 1020 Ala Arg His Glu Asn Glu GlyLeu Val Ser Ser Gly Gln Ser Leu 1025 1030 1035 Tyr Thr Ser Gly Glu LysGlu Ser Asp Ser Ser Ala Ser Thr Ser 1040 1045 1050 Leu Pro Val Glu GluSer Gln Ala Gln Gly Asn Glu Ser Leu Phe 1055 1060 1065 Ser Lys Tyr ThrAsn Ser Lys Ile Pro Tyr Phe Leu Leu Phe Leu 1070 1075 1080 Ile Phe LeuIle Thr Val Tyr His Tyr Asp Leu Met Ile Gly Leu 1085 1090 1095 Thr PheTyr Val Leu Ser Leu Ser Trp Leu Ser Trp Glu Glu Gly 1100 1105 1110 ArgGln Lys Glu Ser Val Lys Lys Lys 1115 1120 3 496 PRT Homo sapiens 3 MetGly Thr Gln Lys Val Thr Pro Ala Leu Ile Phe Ala Ile Thr Val 1 5 10 15Ala Thr Ile Gly Ser Phe Gln Phe Gly Tyr Asn Thr Gly Val Ile Asn 20 25 30Ala Pro Glu Lys Ile Ile Lys Glu Phe Ile Asn Lys Thr Leu Thr Asp 35 40 45Lys Gly Asn Ala Pro Pro Ser Glu Val Leu Leu Thr Ser Leu Trp Ser 50 55 60Leu Ser Val Ala Ile Phe Ser Val Gly Gly Met Ile Gly Ser Phe Ser 65 70 7580 Val Gly Leu Phe Val Asn Arg Phe Gly Arg Arg Asn Ser Met Leu Ile 85 9095 Val Asn Leu Leu Ala Val Thr Gly Gly Cys Phe Met Gly Leu Cys Lys 100105 110 Val Ala Lys Ser Val Glu Met Leu Ile Leu Gly Arg Leu Val Ile Gly115 120 125 Leu Phe Cys Gly Leu Cys Thr Gly Phe Val Pro Met Tyr Ile GlyGlu 130 135 140 Ile Ser Pro Thr Ala Leu Arg Gly Ala Phe Gly Thr Leu AsnGln Leu 145 150 155 160 Gly Ile Val Val Gly Ile Leu Val Ala Gln Ile PheGly Leu Glu Phe 165 170 175 Ile Leu Gly Ser Glu Glu Leu Trp Pro Leu LeuLeu Gly Phe Thr Ile 180 185 190 Leu Pro Ala Ile Leu Gln Ser Ala Ala LeuPro Phe Cys Pro Glu Ser 195 200 205 Pro Arg Phe Leu Leu Ile Asn Arg LysGlu Glu Glu Asn Ala Lys Gln 210 215 220 Ile Leu Gln Arg Leu Trp Gly ThrGln Asp Val Ser Gln Asp Ile Gln 225 230 235 240 Glu Met Lys Asp Glu SerAla Arg Met Ser Gln Glu Lys Gln Val Thr 245 250 255 Val Leu Glu Leu PheArg Val Ser Ser Tyr Arg Gln Pro Ile Ile Ile 260 265 270 Ser Ile Val LeuGln Leu Ser Gln Gln Leu Ser Gly Ile Asn Ala Val 275 280 285 Phe Tyr TyrSer Thr Gly Ile Phe Lys Asp Ala Gly Val Gln Glu Pro 290 295 300 Ile TyrAla Thr Ile Gly Ala Gly Val Val Asn Thr Ile Phe Thr Val 305 310 315 320Val Ser Leu Phe Leu Val Glu Arg Ala Gly Arg Arg Thr Leu His Met 325 330335 Ile Gly Leu Gly Gly Met Ala Phe Cys Ser Thr Leu Met Thr Val Ser 340345 350 Leu Leu Leu Lys Asp Asn Tyr Asn Gly Met Ser Phe Val Cys Ile Gly355 360 365 Ala Ile Leu Val Phe Val Ala Phe Phe Glu Ile Gly Pro Gly ProIle 370 375 380 Pro Trp Phe Ile Val Ala Glu Leu Phe Ser Gln Gly Pro ArgPro Ala 385 390 395 400 Ala Met Ala Val Ala Gly Cys Ser Asn Trp Thr SerAsn Phe Leu Val 405 410 415 Gly Leu Leu Phe Pro Ser Ala Ala His Tyr LeuGly Ala Tyr Val Phe 420 425 430 Ile Ile Phe Thr Gly Phe Leu Ile Thr PheLeu Ala Phe Thr Phe Phe 435 440 445 Lys Val Pro Glu Thr Arg Gly Arg ThrPhe Glu Asp Ile Thr Arg Ala 450 455 460 Phe Glu Gly Gln Ala His Gly AlaAsp Arg Ser Gly Lys Asp Gly Val 465 470 475 480 Met Glu Met Asn Ser IleGlu Pro Ala Lys Glu Thr Thr Thr Asn Val 485 490 495

What is claimed is:
 1. A method for selecting modulators of aninteraction between a first protein and a second protein, (a) said firstprotein being selected from the group consisting of (i) VAP-A (SEQ IDNO:1), (ii) a VAP-A homologue having an amino acid sequence at least 90%identity to that of VAP-A (SEQ ID NO:1) and capable of interacting witha protein selected from the group consisting of PPP1R3 (SEQ ID NO:2) andGTR3 (SEQ ID NO:3), (iii) a VAP-A fragment of VAP-A (SEQ ID NO:1)capable of interacting with a protein selected from the group consistingof PPP1R3 (SEQ ID NO:2) and GTR3 (SEQ ID NO:3), and (iv) a fusionprotein containing VAP-A (SEQ ID NO:1), said VAP-A homologue or saidVAP-A fragment; and (b) said second protein being selected from thegroup consisting of (1) PPP1R3 (SEQ ID NO:2) and GTR3 (SEQ ID NO:3), (2)a homologue of a protein selected from the group consisting of PPP1R3(SEQ ID NO:2) and GTR3 (SEQ ID NO:3) having an amino acid sequence atleast 90% identity to that of PPP1R3 (SEQ ID NO:2) or GTR3 (SEQ ID NO:3)and capable of interacting with VAP-A (SEQ ID NO:1), (3) a PPP1R3fragment of PPP1R3 (SEQ ID NO:2) or a GTR3 fragment of GTR3 (SEQ IDNO:3) and capable of interacting with VAP-A (SEQ ID NO:1), and (4) afusion protein containing a protein selected from the group consistingof PPP1R3 (SEQ ID NO:2) and GTR3 (SEQ ID NO:3), said protein homologueor said PPP1R3 or GTR3 fragment, said method comprising: contacting saidfirst protein with said second protein in the presence of a testcompound; and detecting the interaction between said first protein andsaid second protein; and selecting a test compound that modulates theinteraction between a first protein and a second protein.
 2. The methodof claim 1, wherein at least one of said first and second proteins is afusion protein having a detectable tag.
 3. The method of claim 1,wherein said contacting step is conducted in vitro.
 4. The method ofclaim 1, wherein the interaction between said first protein and saidsecond protein is determined in a host cell.
 5. The method of claim 4,wherein said host cell is a yeast cell.
 6. The method of claim 1,wherein said detecting step comprises measuring the amount of theprotein complex formed by said first and second proteins.
 7. The methodof claim 1, further comprising a step of generating a data set definingone or more selected test compounds, said data set being embodied in atransmittable form.
 8. A method for selecting compounds capable ofinterfering with the interaction between a first protein and a secondprotein, wherein (a) said first protein is selected from the groupconsisting of (i) VAP-A (SEQ ID NO:1), (ii) a VAP-A homologue having anamino acid sequence at least 90% identity to that of VAP-A (SEQ ID NO:1)and capable of interacting with a protein selected from the groupconsisting of PPP1R3 (SEQ ID NO:2) and GTR3 (SEQ ID NO:3), (iii) a VAP-Afragment of VAP-A (SEQ ID NO:1) capable of interacting with a proteinselected from the group consisting of PPP1R3 (SEQ ID NO:2) and GTR3 (SEQID NO:3), and (iv) a fusion protein containing VAP-A (SEQ ID NO:1), saidVAP-A homologue or said VAP-A fragment; and (b) said second protein isselected from the group consisting of (1) PPP1R3 (SEQ ID NO:2) and GTR3(SEQ ID NO:3), (2) a homologue of a protein selected from the groupconsisting of PPP1R3 (SEQ ID NO:2) and GTR3 (SEQ ID NO:3) having anamino acid sequence at least 90% identity to that of PPP1R3 (SEQ IDNO:2) or GTR3 (SEQ ID NO:3) and capable of interacting with VAP-A (SEQID NO:1), (3) a PPP1R3 fragment of PPP1R3 (SEQ ID NO:2) or a GTR3fragment of GTR3 (SEQ ID NO:3) and capable of interacting with VAP-A(SEQ ID NO:1), and (4) a fusion protein containing a protein selectedfrom the group consisting of PPP1R3 (SEQ ID NO:2) and GTR3 (SEQ IDNO:3), said protein homologue or said PPP1R3 or GTR3 fragment, saidmethod comprising: contacting said first protein with said secondprotein in the presence of a test compound and detecting the interactionbetween said first protein and said second protein; and contacting saidfirst protein with said second protein in the absence of said testcompound and detecting the interaction between said first protein andsaid second protein; and selecting a compound that interferes theinteraction between a first protein and a second protein.
 9. The methodof claim 8, wherein said contacting steps are conducted in vitro. 10.The method of claim 8, wherein said contacting steps are conducted in ahost cell.
 11. The method of claim 8, wherein the first protein is afusion protein containing VAP-A (SEQ ID NO:1) or said VAP-A fragment ofVAP-A (SEQ ID NO:1), and said second protein is a fusion proteincontaining a protein selected from the group consisting of PPP1R3 (SEQID NO:2), GTR3 (SEQ ID NO:3), a fragment of PPP1R3 (SEQ ID NO:2), and afragment of GTR3 (SEQ ID NO:3).
 12. The method of claim 8, furthercomprising a step of generating a data set defining one or more selectedtest compounds, said data set being embodied in a transmittable form.